Method for increasing expression of rna-encoded proteins

ABSTRACT

The invention relates to an RNA comprising at least one open reading frame (ORF) and comprising at least one modification, which increases the expression of the encoded peptide or protein. Furthermore, the invention relates to the medical use of such a modified RNA administered to a subject by jet injection. The invention relates further to a pharmaceutical composition and to a kit of parts comprising said modified RNA for administration by jet injection, preferably for use in the field of gene therapy and/or genetic vaccination. Additionally, the invention relates to a method for enhancing the (localized) expression of RNA-encoded peptides or proteins in the dermis or muscle (of a mammal) comprising administering the modified RNA by jet injection. And finally, the invention relates to a method of treatment comprising administering the modified RNA by jet injection to a subject in need thereof.

The present application is a continuation of U.S. application Ser. No.15/048,031, filed Feb. 19, 2016, which is a continuation ofInternational Application No. PCT/EP2014/002300, filed Aug. 21, 2014,which claims priority benefit of European Application No.PCT/EP2013/002512, filed Aug. 21, 2013, the entire text of each of theabove referenced disclosures being specifically incorporated herein byreference.

BACKGROUND OF THE INVENTION

The sequence listing that is contained in the file named“CRVCP0139USC1.txt”, which is 52 KB (as measured in Microsoft Windows®)and was created on Mar. 29, 2019, is filed herewith by electronicsubmission and is incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to an RNA comprising at least one open readingframe (ORF) and comprising at least one modification, which increasesthe expression of the encoded peptide or protein. Furthermore, theinvention relates to the medical use of such a modified RNA administeredto a subject by jet injection. The invention relates further to apharmaceutical composition and to a kit of parts comprising saidmodified RNA for administration by jet injection, preferably for use inthe field of gene therapy and/or genetic vaccination. Additionally, theinvention relates to a method for enhancing the (localized) expressionof RNA-encoded peptides or proteins in the dermis or muscle (of amammal) comprising administering the modified RNA by jet injection. Andfinally, the invention relates to a method of treatment comprisingadministering the modified RNA by jet injection to a subject in needthereof.

BACKGROUND OF THE INVENTION

Gene therapy and genetic vaccination belong to the most promising andquickly developing methods of modern medicine. They may provide highlyspecific and individual options for therapy of a large variety ofdiseases. Particularly, inherited genetic diseases but also autoimmunediseases, infectious diseases, cancerous or tumour-related diseases aswell as inflammatory diseases may be the subject of such treatmentapproaches. Also, it is envisaged to prevent (early) onset of suchdiseases by these approaches.

The main conceptual rational behind gene therapy is appropriatemodulation of impaired gene expression associated with pathologicalconditions of specific diseases. Pathologically altered gene expressionmay result in lack or overproduction of essential gene products, forexample, signalling factors such as hormones, housekeeping factors,metabolic enzymes, structural proteins or the like. Altered geneexpression may not only be due to mis-regulation of transcription and/ortranslation, but also due to mutations within the ORF coding for aparticular protein. Pathological mutations may be caused by e.g.chromosomal aberration, or by more specific mutations, such as pointmutations or frame-shift mutations, all of which may result in limitedfunctionality and, potentially, total loss of function of the geneproduct. However, misregulation of transcription and/or translation mayalso occur, if mutations affect genes encoding proteins, which areinvolved in the transcriptional or translational machinery of the cell.Such mutations may lead to pathological up- or down-regulation of genes,which are—as such—functional. Genes encoding gene products, which exertsuch regulating functions, may be, for instance, transcription factors,signal receptors, messenger proteins or the like. However, loss offunction of such genes encoding regulatory proteins may, under certaincircumstances, be reversed by artificial introduction of other factorsacting further downstream of the impaired gene product. Such genedefects may also be compensated by gene therapy via substitution of theaffected gene itself.

As can be seen from the above, both methods, gene therapy and geneticvaccination, are essentially based on the administration of nucleic acidmolecules to a patient and subsequent transcription and/or translationof the encoded genetic information.

DNA as well as RNA may be used as nucleic acid molecules foradministration in the context of gene therapy or genetic vaccination.DNA is known to be relatively stable and easy to handle. However, theuse of DNA bears the risk of undesired insertion of the administeredDNA-fragments into the patient's genome potentially resulting in loss offunction of the impaired genes. As a further risk, the undesiredgeneration of anti-DNA antibodies has emerged. Another drawback is thelimited expression level of the encoded peptide or protein that can beachieved by DNA administration and its subsequenttranscription/translation. Among other factors, the presence of specifictranscription factors, which regulate DNA transcription, has a majorimpact on the expression level of the administered DNA. In the absenceof such factors, DNA transcription will not yield satisfying amounts ofRNA. As a result, the level of translated peptide or protein obtained islimited.

By using RNA instead of DNA for gene therapy or genetic vaccination, therisk of undesired genomic integration and generation of anti-DNAantibodies is minimized or can be avoided altogether. However, RNA isconsidered to be a rather unstable molecular species. On the one hand,in the extracellular space, RNA is subject to degradation by almostubiquitous RNAses. On the other hand, in vivo mRNA half-life in thecytoplasm is limited by the rate of enzymatic mRNA decay, which depends,at least in part, on cis-acting elements in the mRNA molecule. Thereby,controlled degradation of mRNA contributes to the fine regulation ofeukaryotic gene expression (Friedel et al., Conserved principles ofmammalian transcriptional regulation revealed by RNA half-life, NucleicAcid Research, 2009, 1-12). Accordingly, each naturally occurring mRNAhas its individual half-life depending on the gene, from which the mRNAis derived.

For many years it was generally accepted that mRNA is too unstable to beefficiently used for gene therapy purposes. In the last decade, however,several research groups faced this challenge and not only proved thefeasibility of mRNA-mediated transfection with surprising resultsregarding transfection efficiency and duration of protein expression,but were also able to demonstrate major advantages over the use of pDNA.One of these advantages is the circumstance that mRNA does not need tocross the nuclear barrier for its encoded proteins to be expressed(reviewed in Tavernier et al., J Control Release. 2011 Mar. 30;150(3):238-47. PMID: 20970469).

For gene therapy and genetic vaccination, modified and thereforestabilized RNA is usually more suitable than unmodified RNA, which isusually degraded quickly. On the one hand, the product encoded by theRNA-sequence is desired to accumulate in vivo. On the other hand, theRNA has to maintain its structural and functional integrity whenprepared for a suitable dosage form, in the course of its storage, andwhen administered. Thus, considerable efforts were undertaken to providestable RNA molecules for gene therapy or genetic vaccination in order toprevent them from being subject to early degradation or decay.

After introduction into the cell, the half-life of (non-stabilized) mRNAis limited. As a result, the production of protein encoded by this mRNAlasts for a few days maximally. Obviously, this fact limits theapplicability of (non-stabilized) mRNA-based gene therapy. It cannote.g. be used to correct hereditary diseases, because this would requirerepetitive administrations (reviewed in Tavernier et al., J ControlRelease. 2011 Mar. 30; 150(3):238-47. PMID: 20970469).

As mentioned above, mRNA is generally considered as a fairly unstablemolecule, compared to DNA, especially once it reaches the cytoplasmwhere it is exposed to degrading enzymes. The main reason for itsinstability is the presence of a hydroxyl group on the second carbonatom of the sugar moiety, which, due to sterical hindrance, preventsmRNA from adopting a stable double β-helix structure and which makes themolecule more prone to hydrolytic degradation. Initial reports ofintracellular mRNA delivery were subject to scepticism, mainly becauseof the belief that mRNA is extremely labile and could not withstand thetransfection protocols.

As mentioned above, it is one of the advantages of RNA that it issufficient for its expression to occur to deliver the RNA to thecytoplasm of a cell (in contrast to DNA, which has to cross the nuclearenvelope). However, naked nucleic acid molecules do not enter cellsefficiently because of their large size and hydrophilic nature due tonegatively charged phosphate groups. In addition, they are verysusceptible to nuclease-mediated degradation. Therefore, a challenge forgene therapy is to develop effective and safe means of RNA delivery to acell.

In general, viral and non-viral delivery methods have been described(see, for example, Tavernier et al., J Control Release. 2011 Mar. 30;150(3):238-47. PMID: 20970469). With respect to safety and processeconomy, non-viral methods are usually preferred. While some of thesemethods are based on the physical interruption of the cell membrane'sbarrier function, others employ cationic carrier molecules in order tofacilitate gene transfer to targeted cells without degradation of thedelivered gene.

First insight into the uptake mechanism of naked mRNA was gained by amouse study investigating intradermal administration by injection. Inthis context, it could be shown that local entry into cells of thedermis turned out to be saturable, which means that only a definedprotein level can be reached by intradermal injection of the mRNA. Moreelaborate work in vitro confirmed saturability of uptake anddemonstrated that it is also temperature and dose dependent (reviewed inSchlake et al.; RNA Biology 9:11, 1-12; November 2012).

Thus far, intradermal injection is most frequently used for mRNA-basedapplications, allowing uptake of the mRNA by Langerhans' cells anddermal DCs after which transport to the draining lymph nodes is assumed(reviewed in Van Lint et al.; Human Vaccines & Immunotherapeutics 9:2,248-257; February 2013). Other administration routes that have been usedin order to deliver RNA, comprise intramuscular injection, intranodalinjection (injection into a lymph node) and intratumoral injection.

It is evident not only from experiments using RNA as a vaccine thatprotein expression mediated by introduction of heterologous mRNA in vivois generally possible and sufficient for raising a detectable immuneresponse. However, raising an effective immune response and, even more,achieving a therapeutic effect by mRNA-mediated protein supply may bemore demanding in terms of the required level of protein expression.

The efficient transfer of RNA into the cells of a subject thus stillrepresents a bottleneck in the efficient expression of proteinsintroduced into the cell by means of heterologous RNA. The therapeuticeffectiveness of RNA-based medicaments, particularly in the field ofgene therapy, largely depends on the efficient expression of the geneproducts encoded in the therapeutic RNA molecule.

Therefore it is an object of the invention to improve the expression ofthe genetic information comprised in an RNA molecule, which isintroduced into a cell or a tissue. Specifically, it is the object ofthe invention to improve the expression of a protein encoded in an RNAmolecule, which is used for gene therapy or genetic vaccination andwhich is administered to a subject.

The object underlying the present invention is solved by the claimedsubject matter.

SUMMARY OF THE INVENTION

The present invention provides an RNA comprising at least one openreading frame (ORF) and comprising at least one modification (“modifiedRNA”), which increases the expression of the encoded protein, formedical use, wherein the modified RNA is administered to a subject byjet injection. Specifically, the invention provides a modified RNAcoding for at least one peptide or protein, whose expression is furtherincreased by jet injection. Furthermore the invention provides apharmaceutical composition and a kit of parts comprising said modifiedRNA for administration by jet injection, preferably for use in the fieldof gene therapy and/or genetic vaccination. Additionally, the presentinvention provides a method for increasing protein expression by jetinjection of a respective RNA comprising at least one modification,which increases the expression of the protein encoded in that RNA.

In summary, the object of the present invention is solved by theprovision of a modified RNA, wherein the expression of the encodedprotein in a target tissue or in a target cell is additionally increasedby jet injection.

BRIEF DESCRIPTION OF THE FIGURES

The figures shown in the following are merely illustrative and shalldescribe the present invention in a further way. These figures shall notbe construed to limit the present invention thereto.

FIG. 1: Luciferase expression in guinea pigs

Guinea pigs were injected with 20 μg of modified mRNA encoding Photinuspyralis luciferase (PpLuc(GC)-muag-A64-C30-histoneSL) by jet injectionor conventional intradermal needle injection. 24 h after injection, skinsamples were prepared and luciferase expression was measured.

FIG. 2: Luciferase expression in guinea pigs

Guinea pigs were injected with different doses (10, 40 and 80 μg) ofmodified mRNA encoding Photinus pyralis luciferase(PpLuc(GC)-muag-A64-C30-histoneSL) by jet injection or conventionalintradermal needle injection. 24 h after injection, skin samples wereprepared and luciferase expression was measured.

FIG. 3: Comparison of unmodified and modified mRNA

Guinea pigs were injected with different doses (5 and 80 μg) ofunmodified mRNA (PpLuc(wt)-A30) and modified mRNA(PpLuc(GC)-muag-A64-C30-histoneSL) encoding Photinus pyralis luciferaseby jet injection or conventional intradermal needle injection. 24 hafter injection, skin samples were prepared and luciferase expressionwas measured.

FIG. 4: mRNA sequence R1265: PpLuc(GC)-muag-A64-C30-histoneSL (SEQ IDNO: 46)

FIG. 5: mRNA sequence R2652: PpLuc(wt)-A30 (SEQ ID NO: 47)

FIG. 6: mRNA sequence R3454: PpLuc(wt)-A64 (SEQ ID NO: 48)

FIG. 7: mRNA sequence R2462: PpLuc(GC)-A64-C30-HistoneSL (SEQ ID NO: 49)

FIG. 8: mRNA sequence R1256: PpLuc(GC)-muag-A64-C30 (SEQ ID NO: 50)

FIG. 9: mRNA sequence R2393: PpLuc(nat)-muag-A64-C30-HistoneSL (SEQ IDNO: 51)

FIG. 10: mRNA sequence R2403: RAV-G(GC)-muag-A64-C30-histoneSL (SEQ IDNO: 52)

FIG. 11: mRNA sequence R3513: EPO(wt)-A30 (SEQ ID NO: 53)

FIG. 12: mRNA sequence R3135: HSD17B4-EPO(GC)-albumin7-A64-C30-histoneSL(SEQ ID NO: 54)

FIG. 13: Comparison of unmodified and modified mRNA coding forErythropoetin (EPO)

Guinea pigs were injected with unmodified mRNA (R3513: EPO(wt)-A30) ormodified mRNA (HSD17B4-EPO(GC)-albumin7-A64-C30-histoneSL) encodingErythropoetin (EPO) by jet injection or conventional intradermal needleinjection. Each animal was injected at 3 sites with 80 μg of RNA perinjection site. 24 h after injection, blood was sampled and EPOexpression was measured as described in Example 5.

FIG. 14: G/C optimized mRNA sequence R2510 (SEQ ID NO: 64) coding forRSV-F protein of the RSV long strain (RSV-F long) as comprised in theRSV-F mRNA vaccine.

FIGS. 15A-B: Comparison of conventional syringe-needle injection and jetinjection of an RSV mRNA vaccine (antibody titers).

Female guinea pigs (n=8/group) were intradermally (i.d.) injected withthe RSV-F mRNA vaccine (80 μg of R2510), either 1×100 μl withconventional needle injection (i.d.), 4×25 μl with needle injection(i.d.), or 1×100 μl with jet injection (i.d.). A control group (n=2) wasneedle-injected intramuscularly (i.m.) with 20 μg of inactivated RSVlong (2×50 μl). All animals received boost injections on days 14 and 28.Blood samples were collected on day −3 (three days before the firstvaccination) and on days 7, 21 and 42 for the determination of anti-RSVF antibody titers. The experiment was performed as described in Example4.

FIG. 15 A:IgG1 endpoint titers determined by ELISA.

FIG. 15 B: IgG2a endpoint titers as determined by ELISA.

As can be seen, the RSV-F mRNA vaccine already induced anti-F proteinantibodies of the IgG subclass (A) and the IgG2a subclass (B) on day 21(one week after the first boost vaccination on day 14) when the vaccinewas administered by jet injection (1×100 al). Comparable antibody titerswere only reached on day 42 (two weeks after the second boostvaccination on day 28) when the vaccine was administered by conventionalneedle injection (4×25 al).

FIG. 16: Comparison of conventional syringe-needle injection and jetinjection of an RSV mRNA vaccine (virus neutralization titers).

The experiment was performed as described in Example 4. Virusneutralizing titers (VNTs) were determined by plaque reduction assay ondays −3, 21 and 42. Median VNTs are plotted over time. VNTs areindicated as reciprocal neutralizing antibody titers at 60% reductionend-point of the virus control. As can be seen, significant RSVneutralization titers were measured on day 42 only when the vaccine wasadministered by jet injection (1×100 al).

For the sake of clarity and readability the following definitions areprovided. Any technical feature mentioned for these definitions may beread on each and every embodiment of the invention. Additionaldefinitions and explanations may be specifically provided in the contextof these embodiments. Where nucleic acid sequences are reported in thecontext of the present invention, these sequences generally compriseboth, the specific RNA or DNA sequence as well as its corresponding DNAor RNA counterpart, respectively. For example, where a DNA sequence isprovided, the skilled person knows that the corresponding RNA sequenceis obtained by exchange of thymine by uracil residues and vice versa.

Gene Therapy:

Gene therapy may typically be understood to mean a treatment of apatient's body or isolated elements of a patient's body, for exampleisolated tissues/cells, by nucleic acids encoding a peptide or protein.It typically may comprise at least one of the steps of a) administrationof a nucleic acid, preferably an RNA molecule as defined herein,directly to the patient—by whatever administration route—or in vitro toisolated cells/tissues of the patient, which results in transfection ofthe patient's cells either in vivo/ex vivo or in vitro; b) transcriptionand/or translation of the introduced nucleic acid molecule; andoptionally c) re-administration of isolated, transfected cells to thepatient, if the nucleic acid has not been administered directly to thepatient.

Genetic Vaccination:

Genetic vaccination may typically be understood to be vaccination byadministration of a nucleic acid molecule encoding an antigen or animmunogen or fragments thereof. The nucleic acid molecule may beadministered to a subject's body or to isolated cells of a subject. Upontransfection of certain cells of the body or upon transfection of theisolated cells, the antigen or immunogen may be expressed by those cellsand subsequently presented to the immune system, eliciting an adaptive,i.e. antigen-specific immune response. Accordingly, genetic vaccinationtypically comprises at least one of the steps of a) administration of anucleic acid, preferably an RNA molecule as defined herein, to asubject, preferably a patient, or to isolated cells of a subject,preferably a patient, which usually results in transfection of thesubject's cells either in vivo or in vitro; b) transcription and/ortranslation of the introduced nucleic acid molecule; and optionally c)re-administration of isolated, transfected cells to the subject,preferably the patient, if the nucleic acid has not been administereddirectly to the patient.

Immune System:

The immune system may protect organisms from infection. If a pathogensucceeds in passing a physical barrier of an organism and enters thisorganism, the innate immune system provides an immediate, butnon-specific response. If pathogens evade this innate response,vertebrates possess a second layer of protection, the adaptive immunesystem. Here, the immune system adapts its response during an infectionto improve its recognition of the pathogen. This improved response isthen retained after the pathogen has been eliminated, in the form of animmunological memory, and allows the adaptive immune system to mountfaster and stronger attacks each time this pathogen is encountered.According to this, the immune system comprises the innate and theadaptive immune system. Each of these two parts typically containsso-called humoral and cellular components.

Adaptive Immune System:

The adaptive immune system is essentially dedicated to eliminate orprevent pathogenic growth. It typically regulates the adaptive immuneresponse by providing the vertebrate immune system with the ability torecognize and remember specific pathogens (to generate immunity), and tomount stronger attacks each time the pathogen is encountered. The systemis highly adaptable because of somatic hypermutation (a process ofaccelerated somatic mutations), and V(D)J recombination (an irreversiblegenetic recombination of antigen receptor gene segments). This mechanismallows a small number of genes to generate a vast number of differentantigen receptors, which are then uniquely expressed on each individuallymphocyte. Because the gene rearrangement leads to an irreversiblechange in the DNA of each cell, all of the progeny (offspring) of such acell will then inherit genes encoding the same receptor specificity,including the Memory B cells and Memory T cells that are the keys tolong-lived specific immunity.

Innate Immune System:

The innate immune system, also known as non-specific (or unspecific)immune system, typically comprises the cells and mechanisms that defendthe host from infection by other organisms in a non-specific manner.This means that the cells of the innate system may recognize and respondto pathogens in a generic way, but unlike the adaptive immune system, itdoes not confer long-lasting or protective immunity to the host. Theinnate immune system may be, e.g., activated by ligands of Toll-likereceptors (TLRs) or other auxiliary substances such aslipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines,lymphokines, interleukins or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26,IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta,IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, andhGH, a ligand of human Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, a ligand of murine Toll-like receptorTLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11,TLR12 or TLR13, a ligand of a NOD-like receptor, a ligand of a RIG-Ilike receptor, an immunostimulatory nucleic acid, an immunostimulatoryRNA (isRNA), a CpG-DNA, an antibacterial agent, or an anti-viral agent.The pharmaceutical composition according to the present invention maycomprise one or more such substances. Typically, a response of theinnate immune system includes recruiting immune cells to sites ofinfection, through the production of chemical factors, includingspecialized chemical mediators, called cytokines; activation of thecomplement cascade; identification and removal of foreign substancespresent in organs, tissues, the blood and lymph, by specialized whiteblood cells; activation of the adaptive immune system; and/or acting asa physical and chemical barrier to infectious agents.

Immune Response:

An immune response may typically be a specific reaction of the adaptiveimmune system to a particular antigen (so-called specific or adaptiveimmune response) or an unspecific reaction of the innate immune system(so-called unspecific or innate immune response), or a combinationthereof.

Adaptive Immune Response:

The adaptive immune response is typically understood to be anantigen-specific response of the immune system. Antigen specificityallows for the generation of responses that are tailored to specificpathogens or pathogen-infected cells. The ability to mount thesetailored responses is usually maintained in the body by “memory cells”.Should a pathogen infect the body more than once, these specific memorycells are used to quickly eliminate it. In this context, the first stepof an adaptive immune response is the activation of naïveantigen-specific T cells or different immune cells able to induce anantigen-specific immune response by antigen-presenting cells. Thisoccurs in the lymphoid tissues and organs, through which naïve T cellsare constantly passing. The three cell types that may serve asantigen-presenting cells are dendritic cells, macrophages, and B cells.Each of these cells has a distinct function in eliciting immuneresponses. Dendritic cells may take up antigens by phagocytosis andmacropinocytosis and may become stimulated by contact with e.g. aforeign antigen to migrate to the local lymphoid tissue, where theydifferentiate into mature dendritic cells. Macrophages ingestparticulate antigens such as bacteria and are induced by infectiousagents or other appropriate stimuli to express MHC molecules. The uniqueability of B cells to bind and internalize soluble protein antigens viatheir receptors may also be important to induce T cells. MHC-moleculesare, typically, responsible for presentation of an antigen to T-cells.Therein, presenting the antigen on MHC molecules leads to activation ofT cells which induces their proliferation and differentiation into armedeffector T cells. The most important function of effector T cells is thekilling of infected cells by CD8+ cytotoxic T cells and the activationof macrophages by Th1 cells which together make up cell-mediatedimmunity, and the activation of B cells by both Th2 and Th1 cells toproduce different classes of antibody, thus driving the humoral immuneresponse. T cells recognize an antigen by their T cell receptors whichdo not recognize and bind the antigen directly, but instead recognizeshort peptide fragments e.g. of pathogen-derived protein antigens, e.g.so-called epitopes, which are bound to MHC molecules on the surfaces ofother cells.

Cellular Immunity/Cellular Immune Response:

Cellular immunity relates typically to the activation of macrophages,natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, andthe release of various cytokines in response to an antigen. In moregeneral terms, cellular immunity is not based on antibodies, but on theactivation of cells of the immune system. Typically, a cellular immuneresponse may be characterized e.g. by activating antigen-specificcytotoxic T-lymphocytes that are able to induce apoptosis in cells, e.g.specific immune cells like dendritic cells or other cells, displayingepitopes of foreign antigens on their surface. Such cells may bevirus-infected or infected with intracellular bacteria, or cancer cellsdisplaying tumor antigens. Further characteristics may be activation ofmacrophages and natural killer cells, enabling them to destroy pathogensand stimulation of cells to secrete a variety of cytokines thatinfluence the function of other cells involved in adaptive immuneresponses and innate immune responses.

Immunogen:

In the context of the present invention an immunogen may be typicallyunderstood to be a compound that is able to stimulate an immuneresponse. Preferably, an immunogen is a peptide, polypeptide, orprotein. In a particularly preferred embodiment, an immunogen in thesense of the present invention is the product of translation of aprovided nucleic acid molecule, preferably an artificial nucleic acidmolecule as defined herein. Typically, an immunogen elicits at least anadaptive immune response.

Antigen:

In the context of the present invention “antigen” refers typically to asubstance, which may be recognized by the immune system, preferably bythe adaptive immune system, and is capable of triggering anantigen-specific immune response, e.g. by formation of antibodies and/orantigen-specific T cells as part of an adaptive immune response.Typically, an antigen may be or may comprise a peptide or protein whichmay be presented by the MHC to T-cells.

Epitope:

Epitopes (also called ‘antigen determinant’) can be distinguished in Tcell epitopes and B cell epitopes. T cell epitopes or parts of theproteins in the context of the present invention may comprise fragmentspreferably having a length of about 6 to about 20 or even more aminoacids, e.g. fragments as processed and presented by MHC class Imolecules, preferably having a length of about 8 to about 10 aminoacids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragmentsas processed and presented by MHC class II molecules, preferably havinga length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18,19, 20 or even more amino acids, wherein these fragments may be selectedfrom any part of the amino acid sequence.

These fragments are typically recognized by T cells in form of a complexconsisting of the peptide fragment and an MHC molecule, i.e. thefragments are typically not recognized in their native form. B cellepitopes are typically fragments located on the outer surface of(native) protein or peptide antigens as defined herein, preferablyhaving 5 to 15 amino acids, more preferably having 5 to 12 amino acids,even more preferably having 6 to 9 amino acids, which may be recognizedby antibodies, i.e. in their native form.

Such epitopes of proteins or peptides may furthermore be selected fromany of the herein mentioned variants of such proteins or peptides. Inthis context, antigenic determinants can be conformational ordiscontinuous epitopes, which are composed of segments of the proteinsor peptides as defined herein that are discontinuous in the amino acidsequence of the proteins or peptides as defined herein, but are broughttogether in the three-dimensional structure or continuous or linearepitopes, which are composed of a single polypeptide chain.

Adjuvant/Adjuvant Component:

An adjuvant or an adjuvant component in the broadest sense is typicallya pharmacological and/or immunological agent that may modify, e.g.enhance, the effect of other agents, such as a drug or vaccine. It is tobe interpreted in a broad sense and refers to a broad spectrum ofsubstances. Typically, these substances are able to increase theimmunogenicity of antigens. For example, adjuvants may be recognized bythe innate immune systems and, e.g., may elicit an innate immuneresponse. “Adjuvants” typically do not elicit an adaptive immuneresponse. Insofar, “adjuvants” do not qualify as antigens. Their mode ofaction is distinct from the effects triggered by antigens resulting inan adaptive immune response.

Protein:

A protein typically comprises one or more peptides or polypeptides. Aprotein is typically folded into 3-dimensional form, which may berequired for the protein to exert its biological function.

Peptide:

A peptide or polypeptide is typically a polymer of amino acid monomers,linked by peptide bonds. It typically contains less than 50 monomerunits. Nevertheless, the term peptide is not a disclaimer for moleculeshaving more than 50 monomer units. Long peptides are also calledpolypeptides, typically having between 50 and 600 monomeric units.

Fragment or Part of a Protein:

Fragments or parts of a protein in the context of the present inventionare typically understood to be peptides corresponding to a continuouspart of the amino acid sequence of a protein, preferably having a lengthof about 6 to about 20 or even more amino acids, e.g. parts as processedand presented by MHC class I molecules, preferably having a length ofabout 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 11, or 12amino acids), or fragments as processed and presented by MHC class IImolecules, preferably having a length of about 13 or more amino acids,e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, whereinthese fragments may be selected from any part of the amino acidsequence. These fragments are typically recognized by T cells in form ofa complex consisting of the peptide fragment and an MHC molecule, i.e.the fragments are typically not recognized in their native form.Fragments or parts of the proteins as defined herein may also compriseepitopes or functional sites of those proteins. Preferably, fragments orparts of a proteins in the context of the invention are antigens,particularly immunogens, e.g. antigen determinants (also called‘epitopes’), or do have antigenic characteristics, eliciting an adaptiveimmune response. Therefore, fragments of proteins or peptides maycomprise at least one epitope of those proteins or peptides.Furthermore, also domains of a protein, like the extracellular domain,the intracellular domain or the transmembrane domain, and shortened ortruncated versions of a protein may be understood to comprise a fragmentof a protein.

Pharmaceutically Effective Amount:

A pharmaceutically effective amount in the context of the invention istypically understood to be an amount that is sufficient to induce apharmaceutical effect, such as an immune response, altering apathological level of an expressed peptide or protein, or substituting alacking gene product, e.g., in case of a pathological situation.

Sequence Identity:

Two or more sequences are identical if they exhibit the same length andorder of nucleotides or amino acids. The percentage of identitytypically describes the extent, to which two sequences are identical,i.e. it typically describes the percentage of nucleotides thatcorrespond in their sequence position with identical nucleotides of areference sequence. For determination of the degree of identity, thesequences to be compared are considered to exhibit the same length, i.e.the length of the longest sequence of the sequences to be compared. Thismeans that a first sequence consisting of 8 nucleotides is 80% identicalto a second sequence consisting of 10 nucleotides comprising the firstsequence. In other words, in the context of the present invention,identity of sequences preferably relates to the percentage ofnucleotides of a sequence which have the same position in two or moresequences having the same length. Gaps are usually regarded asnon-identical positions, irrespective of their actual position in analignment.

Fragment of a Sequence:

A fragment of a sequence may typically be a shorter portion of afull-length sequence of e.g. a nucleic acid molecule or an amino acidsequence. Accordingly, a fragment, typically, consists of a sequencethat is identical to the corresponding stretch within the full-lengthsequence. A preferred fragment of a sequence in the context of thepresent invention, consists of a continuous stretch of entities, such asnucleotides or amino acids corresponding to a continuous stretch ofentities in the molecule the fragment is derived from, which representsat least 30%, more preferably at least 40%, more preferably at least50%, even more preferably at least 60%, even more preferably at least70%, and most preferably at least 80% of the total (i.e. full-length)molecule, from which the fragment is derived.

Sequence of a Nucleic Acid Molecule:

The sequence of a nucleic acid molecule is typically understood to bethe particular and individual order, i.e. the succession, of itsnucleotides. The sequence of a protein or peptide is typicallyunderstood to be the order, i.e. the succession, of its amino acids.

Stabilized Nucleic Acid Molecule:

A stabilized nucleic acid molecule is a nucleic acid molecule,preferably a DNA or RNA molecule that is modified such, that it is morestable to disintegration or degradation, e.g., by environmental factorsor enzymatic digest, such as by an exo- or endonuclease degradation,than the nucleic acid molecule without the modification. Preferably, astabilized nucleic acid molecule in the context of the present inventionis stabilized in a cell, such as a prokaryotic or eukaryotic cell,preferably in a mammalian cell, such as a human cell. The stabilizationeffect may also be exerted outside of cells, e.g. in a buffer solutionetc., for example, in a manufacturing process for a pharmaceuticalcomposition comprising the stabilized nucleic acid molecule.

Heterologous Sequence:

Two sequences are typically understood to be ‘heterologous’ if they arenot derivable from the same gene. I.e., although heterologous sequencesmay be derivable from the same organism, they naturally (in nature) donot occur in the same nucleic acid molecule, such as in the same mRNA.

Nucleic Acid Molecule:

A nucleic acid molecule is a molecule comprising, preferably consistingof, nucleic acid components. The term nucleic acid molecule preferablyrefers to DNA or RNA molecules. It is preferably used synonymously withthe term “polynucleotide”. Preferably, a nucleic acid molecule is apolymer comprising or consisting of nucleotide monomers, which arecovalently linked to each other by phosphodiester bonds of asugar/phosphate-backbone.

Open Reading Frame:

An open reading frame (ORF) in the context of the invention maytypically be a sequence of several nucleotide triplets, which may betranslated into a peptide or protein. An open reading frame preferablycontains a start codon, i.e. a combination of three subsequentnucleotides coding usually for the amino acid methionine (ATG or AUG),at its 5′-end and a subsequent region, which usually exhibits a length,which is a multiple of 3 nucleotides. An ORF is preferably terminated bya stop-codon (e.g., TAA, TAG, TGA). Typically, this is the onlystop-codon of the open reading frame. Thus, an open reading frame in thecontext of the present invention is preferably a nucleotide sequence,consisting of a number of nucleotides that may be divided by three,which starts with a start codon (e.g. ATG or AUG) and which preferablyterminates with a stop codon (e.g., TAA, TGA, or TAG or UAA, UAG, UGA,respectively). The open reading frame may be isolated or it may beincorporated in a longer nucleic acid sequence, for example in a vectoror an mRNA. An open reading frame may also be termed ‘protein codingregion’.

DNA:

DNA is the usual abbreviation for deoxy-ribonucleic-acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotides. Thesenucleotides are usually deoxy-adenosine-monophosphate,deoxy-thymidine-monophosphate, deoxy-guanosine-monophosphate anddeoxy-cytidine-monophosphate monomers which are—by themselves—composedof a sugar moiety (deoxyribose), a base moiety and a phosphate moiety,and polymerise by a characteristic backbone structure. The backbonestructure is, typically, formed by phosphodiester bonds between thesugar moiety of the nucleotide, i.e. deoxyribose, of a first and aphosphate moiety of a second, adjacent monomer. The specific order ofthe monomers, i.e. the order of the bases linked to thesugar/phosphate-backbone, is called the DNA-sequence. DNA may be singlestranded or double stranded. In the double stranded form, thenucleotides of the first strand typically hybridize with the nucleotidesof the second strand, e.g. by A/T-base-pairing and G/C-base-pairing.

RNA, mRNA:

RNA is the usual abbreviation for ribonucleic-acid. It is a nucleic acidmolecule, i.e. a polymer consisting of nucleotides. These nucleotidesare usually adenosine-monophosphate, uridine-monophosphate,guanosine-monophosphate and cytidine-monophosphate monomers, which areconnected to each other along a so-called backbone. The backbone isformed by phosphodiester bonds between the sugar, i.e. ribose, of afirst and a phosphate moiety of a second, adjacent monomer. The specificsuccession of the monomers is called the RNA-sequence. Usually RNA maybe obtainable by transcription of a DNA-sequence, e.g., inside a cell.In eukaryotic cells, transcription is typically performed inside thenucleus or the mitochondria. In vivo, transcription of DNA usuallyresults in the so-called premature RNA which has to be processed intoso-called messenger-RNA, usually abbreviated as mRNA. Processing of thepremature RNA, e.g. in eukaryotic organisms, comprises a variety ofdifferent posttranscriptional-modifications such as splicing,5′-capping, polyadenylation, export from the nucleus or the mitochondriaand the like. The sum of these processes is also called maturation ofRNA. The mature messenger RNA usually provides the nucleotide sequencethat may be translated into an amino acid sequence of a particularpeptide or protein. Typically, a mature mRNA comprises a 5′-cap,optionally a 5′UTR, an open reading frame, optionally a 3′UTR and apoly(A) sequence. Aside from messenger RNA, several non-coding types ofRNA exist which may be involved in regulation of transcription and/ortranslation. The term “RNA” further encompass other coding RNAmolecules, such as viral RNA, retroviral RNA and replicon RNA.

Bicistronic RNA, Multicistronic RNA:

A bicistronic or multicistronic RNA is typically an RNA, preferably anmRNA, that typically may have two (bicistronic) or more (multicistronic)open reading frames (ORF). An open reading frame in this context is asequence of codons that is translatable into a peptide or protein.

G/C Modified:

A G/C-modified nucleic acid may typically be a nucleic acid, preferablyan RNA molecule as defined herein, based on a modified wild-typesequence comprising a preferably increased number of guanosine and/orcytosine nucleotides as compared to the wild-type sequence. Such anincreased number may be generated by substitution of codons containingadenosine or thymidine nucleotides by codons containing guanosine orcytosine nucleotides. If the enriched G/C content occurs in a codingregion of DNA or RNA, it makes use of the degeneracy of the geneticcode. Accordingly, the codon substitutions preferably do not alter theencoded amino acid residues, but exclusively increase the G/C content ofthe nucleic acid molecule.

5′-cap:

A 5′-cap is an entity, typically a modified nucleotide entity, whichgenerally ‘caps’ the 5′-end of a mature mRNA. A 5′-cap may typically beformed by a modified nucleotide, particularly by a derivative of aguanine nucleotide. Preferably, the 5′-cap is linked to the 5′-terminusvia a 5′-5′-triphosphate linkage. A 5′-cap may be methylated, e.g.m7GpppN, wherein N is the terminal 5′ nucleotide of the nucleic acidcarrying the 5′-cap, typically the 5′-end of an RNA. The naturallyoccurring 5′-CAP is m7GpppN.

Immunostimulatory RNA:

An immunostimulatory RNA (isRNA) in the context of the invention maytypically be an RNA that is able to induce an innate immune response. Itusually does not have an open reading frame and thus does not provide apeptide-antigen or immunogen but elicits an immune response, e.g. bybinding to a specific kind of Toll-like-receptor (TLR) or other suitablereceptors. However, of course also mRNAs having an open reading frameand coding for a peptide/protein may induce an innate immune responseand, thus, may be immunostimulatory RNAs.

Poly(A) Sequence:

A poly(A) sequence, also called poly(A) tail or 3′-poly(A) tail, istypically understood to be a sequence of adenine nucleotides, e.g., ofup to about 400 adenine nucleotides, e.g. from about 20 to about 400,preferably from about 50 to about 400, more preferably from about 50 toabout 300, even more preferably from about 50 to about 250, mostpreferably from about 60 to about 250 adenine nucleotides. A poly(A)sequence is typically located at the 3′end of an mRNA. In the context ofthe present invention, a poly(A) sequence may be located within an mRNAor any other nucleic acid molecule, such as, e.g., in a vector, forexample, in a vector serving as template for the generation of an RNA,preferably an mRNA, e.g., by transcription of the vector.

Polyadenylation:

Polyadenylation is typically understood to be the addition of a poly(A)sequence to a nucleic acid molecule, such as an RNA molecule, e.g. to apremature mRNA. Polyadenylation may be induced by a so-calledpolvadenylation signal. This signal is preferably located within astretch of nucleotides at the 3′-end of a nucleic acid molecule, such asan RNA molecule, to be polyadenylated. A polyadenylation signaltypically comprises a hexamer consisting of adenine and uracil/thyminenucleotides, preferably the hexamer sequence AAUAAA. Other sequences,preferably hexamer sequences, are also conceivable. Polyadenylationtypically occurs during processing of a pre-mRNA (also calledpremature-mRNA). Typically, RNA maturation (from pre-mRNA to maturemRNA) comprises the step of polyadenylation.

3′-Untranslated Region (3′UTR):

A 3′UTR is typically the part of an mRNA which is located between theprotein coding region (i.e. the open reading frame) and the poly(A)sequence of the mRNA. A 3′UTR of the mRNA is not translated into anamino acid sequence. The 3′UTR sequence is generally encoded by thegene, which is transcribed into the respective mRNA during the geneexpression process. The genomic sequence is first transcribed intopre-mature mRNA, which comprises optional introns. The pre-mature mRNAis then further processed into mature mRNA in a maturation process. Thismaturation process comprises the steps of 5′capping, splicing thepre-mature mRNA to excise optional introns and modifications of the3′-end, such as polyadenylation of the 3′-end of the pre-mature mRNA andoptional endo-/or exonuclease cleavages etc. In the context of thepresent invention, a 3′UTR corresponds to the sequence of a mature mRNA,which is located 3′ to the stop codon of the protein coding region,preferably immediately 3′ to the stop codon of the protein codingregion, and which extends to the 5′-side of the poly(A) sequence,preferably to the nucleotide immediately 5′ to the poly(A) sequence. Theterm “corresponds to” means that the 3′UTR sequence may be an RNAsequence, such as in the mRNA sequence used for defining the 3′UTRsequence, or a DNA sequence, which corresponds to such RNA sequence. Inthe context of the present invention, the term “a 3′UTR of a gene”, suchas “3′UTR of alpha or beta globine”, is the sequence, which correspondsto the 3′UTR of the mature mRNA derived from this gene, i.e. the mRNAobtained by transcription of the gene and maturation of the pre-maturemRNA. The term “3′UTR of a gene” encompasses the DNA sequence and theRNA sequence of the 3′UTR.

5′-Untranslated Region (5′UTR):

A 5′-UTR is typically understood to be a particular section of messengerRNA (mRNA). It is located 5′ of the open reading frame of the mRNA.Typically, the 5′UTR starts with the transcriptional start site and endsone nucleotide before the start codon of the open reading frame. The5′-UTR may comprise elements for controlling gene expression, alsocalled regulatory elements. Such regulatory elements may be, forexample, ribosomal binding sites or a 5′-Terminal Oligopyrimidine Tract.The 5′UTR may be posttranscriptionally modified, for example by additionof a 5′-cap. In the context of the present invention, a 5′UTRcorresponds to the sequence of a mature mRNA which is located betweenthe 5′cap and the start codon. Preferably, the 5′UTR corresponds to thesequence, which extends from a nucleotide located 3′ to the 5′-cap,preferably from the nucleotide located immediately 3′ to the 5′cap, to anucleotide located 5′ to the start codon of the protein coding region,preferably to the nucleotide located immediately 5′ to the start codonof the protein coding region. The nucleotide located immediately 3′ tothe 5′cap of a mature mRNA typically corresponds to the transcriptionalstart site. The term “corresponds to” means that the 5′UTR sequence maybe an RNA sequence, such as in the mRNA sequence used for defining the5′UTR sequence, or a DNA sequence which corresponds to such RNAsequence. In the context of the present invention, the term “a 5′UTR ofa gene”, is the sequence, which corresponds to the 5′UTR of the maturemRNA derived from this gene, i.e. the mRNA obtained by transcription ofthe gene and maturation of the pre-mature mRNA. The term “5′UTR of agene” encompasses the DNA sequence and the RNA sequence of the 5′UTR.

5′Terminal Oligopyrimidine Tract (TOP): The 5′terminal oligopyrimidinetract (TOP) is typically a stretch of pyrimidine nucleotides located atthe 5′ terminal region of a nucleic acid molecule, such as the 5′terminal region of certain mRNA molecules or the 5′ terminal region of afunctional entity, e.g. the transcribed region, of certain genes. Thesequence starts with a cytidine, which usually corresponds to thetranscriptional start site, and is followed by a stretch of usuallyabout 3 to 30 pyrimidine nucleotides. For example, the TOP may comprise3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or even more nucleotides. The pyrimidinestretch and thus the 5′ TOP ends one nucleotide 5′ to the first purinenucleotide located downstream of the TOP. Messenger RNA that contains a5′terminal oligopyrimidine tract is often referred to as TOP mRNA.Accordingly, genes that provide such messenger RNAs are referred to asTOP genes. TOP sequences have, for example, been found in genes andmRNAs encoding peptide elongation factors and ribosomal proteins.

TOP motif: In the context of the present invention, a TOP motif is anucleic acid sequence, which corresponds to a 5′TOP as defined above.Thus, a TOP motif in the context of the present invention is preferablya stretch of pyrimidine nucleotides having a length of 3-30 nucleotides.Preferably, the TOP-motif consists of at least 3 pyrimidine nucleotides,preferably at least 4 pyrimidine nucleotides, preferably at least 5pyrimidine nucleotides, more preferably at least 6 nucleotides, morepreferably at least 7 nucleotides, most preferably at least 8 pyrimidinenucleotides, wherein the stretch of pyrimidine nucleotides preferablystarts at its 5′end with a cytosine nucleotide. In TOP genes and TOPmRNAs, the TOP-motif preferably starts at its 5′end with thetranscriptional start site and ends one nucleotide 5′ to the first purinresidue in said gene or mRNA. A TOP motif in the sense of the presentinvention is preferably located at the 5′end of a sequence, whichrepresents a 5′UTR, or at the 5′end of a sequence, which codes for a5′UTR. Thus, preferably, a stretch of 3 or more pyrimidine nucleotidesis called “TOP motif” in the sense of the present invention if thisstretch is located at the 5′end of a respective sequence, such as themodified RNA according to the invention, the 5′UTR element of themodified RNA according to the invention, or the nucleic acid sequence,which is derived from the 5′UTR of a TOP gene as described herein. Inother words, a stretch of 3 or more pyrimidine nucleotides, which is notlocated at the 5′-end of a 5′UTR or a 5′UTR element, but anywhere withina 5′UTR or a 5′UTR element is preferably not referred to as “TOP motif”.

Top Gene:

TOP genes are typically characterised by the presence of a 5′ terminaloligopyrimidine tract. Furthermore, most TOP genes are characterized bya growth-associated translational regulation. However, also TOP geneswith a tissue specific translational regulation are known. As definedabove, the 5′UTR of a TOP gene corresponds to the sequence of a 5′UTR ofa mature mRNA derived from a TOP gene, which preferably extends from thenucleotide located 3′ to the 5′-CAP to the nucleotide located 5′ to thestart codon. A 5′UTR of a TOP gene typically does not comprise any startcodons, preferably no upstream AUGs (uAUGs) or upstream open readingframes (uORFs). Therein, upstream AUGs and upstream open reading framesare typically understood to be AUGs and open reading frames that occur5′ of the start codon (AUG) of the open reading frame that should betranslated. The 5′UTRs of TOP genes are generally rather short. Thelengths of 5′UTRs of TOP genes may vary between 20 nucleotides up to 500nucleotides, and are typically less than about 200 nucleotides,preferably less than about 150 nucleotides, more preferably less thanabout 100 nucleotides. Exemplary 5′UTRs of TOP genes in the sense of thepresent invention are the nucleic acid sequences extending from thenucleotide at position 5 to the nucleotide located immediately 5′ to thestart codon (e.g. the ATG) in the sequences according to SEQ ID Nos.1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 14221-1363 ofthe patent application PCT/EP2012/002448WO2013/143700 or homologs orvariants thereof, whose disclosure is incorporated herewith byreference. In this context, a particularly preferred fragment of a 5′UTRof a TOP gene is a 5′UTR of a TOP gene lacking the 5′TOP motif. The term‘5′UTR of a TOP gene’ preferably refers to the 5′UTR of a naturallyoccurring TOP gene.

Unmodified Reference RNA:

RNA corresponding to the wild type RNA sequence as it is present innature. For example, an unmodified reference mRNA corresponds to an mRNAsequence comprising naturally occuring nucleotides, includingnucleotides comprising naturally occuring modifications, and/ornaturally occuring untranslated regions, such as the naturally occuring5′-CAP structure m7GpppN, optionally the naturally occuring 5′-UTR, ifpresent in the naturally occuring mRNA sequence, the wild type openreading frame, optionally the naturally occuring 3′-UTR if present inthe naturally occuring mRNA sequence and a poly A sequence withapproximately 30 adenosines.

Transfection:

The term ‘transfection’ refers to the introduction of nucleic acidmolecules, such as DNA or RNA (e.g. mRNA) molecules, into cells,preferably into eukaryotic cells. In the context of the presentinvention, the term ‘transfection’ encompasses any method known to theskilled person for introducing nucleic acid molecules into cells,preferably into eukaryotic cells, such as into mammalian cells. Suchmethods encompass, for example, electroporation, lipofection, e.g. basedon cationic lipids and/or liposomes, calcium phosphate precipitation,nanoparticle based transfection, virus based transfection, ortransfection based on cationic polymers, such as DEAE-dextran orpolyethylenimine etc. Preferably, the introduction is non-viral.

Carrier/Polymeric Carrier:

A carrier in the context of the invention may typically be a compoundthat facilitates transport and/or complexation of another compound(cargo). A polymeric carrier is typically a carrier that is formed of apolymer. A carrier may be associated to its cargo by covalent ornon-covalent interaction. A carrier may transport nucleic acids, e.g.RNA or DNA, to the target cells. The carrier may—for some embodiments—bea cationic component.

Cationic Component:

The term “cationic component” typically refers to a charged molecule,which is positively charged (cation) at a pH value typically from 1 to9, preferably at a pH value of or below 9 (e.g. from 5 to 9), of orbelow 8 (e.g. from 5 to 8), of or below 7 (e.g. from 5 to 7), mostpreferably at a physiological pH, e.g. from 7.3 to 7.4. Accordingly, acationic component may be any positively charged compound or polymer,preferably a cationic peptide or protein which is positively chargedunder physiological conditions, particularly under physiologicalconditions in vivo. A ‘cationic peptide or protein’ may contain at leastone positively charged amino acid, or more than one positively chargedamino acid, e.g. selected from Arg, His, Lys or Orn. Accordingly,‘polycationic’ components are also within the scope exhibiting more thanone positive charge under the conditions given.

Vaccine:

A vaccine is typically understood to be a prophylactic or therapeuticmaterial providing at least one antigen, preferably an immunogen. Theantigen or immunogen may be derived from any material that is suitablefor vaccination. For example, the antigen or immunogen may be derivedfrom a pathogen, such as from bacteria or virus particles etc., or froma tumor or cancerous tissue. The antigen or immunogen stimulates thebody's adaptive immune system to provide an adaptive immune response.

Vehicle:

A vehicle is typically understood to be a material that is suitable forstoring, transporting, and/or administering a compound, such as apharmaceutically active compound. For example, it may be aphysiologically acceptable liquid which is suitable for storing,transporting, and/or administering a pharmaceutically active compound.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides a modified ribonucleicacid (RNA) comprising at least one open reading frame (ORF) coding forat least one peptide or protein, wherein the RNA comprises at least onemodification, which increases the expression of its encoded protein orpeptide. The expression of said encoded protein or peptide is furtherincreased by administration of the modified RNA by jet injection. Inthis context the increase in protein production due to the at least onemodification of the RNA is referred to herein as“modification-associated increase of protein expression”.

The increase in protein production due to jet injection of the RNA isreferred to herein as “injection-associated increase of proteinexpression”, independently if modified RNA or an unmodified referenceRNA is used.

The term ‘modification-associated increase of protein expression’ eitherrefers to a situation, where the total amount of protein that isproduced from the modified RNA measured for a specific time period isincreased in comparison to the total amount of protein produced from anunmodified reference RNA lacking the modification or to a situation,where the protein level that is produced from the modified RNA measuredfor a specific time point is increased in comparison to the proteinlevel produced from an unmodified reference RNA lacking themodification.

The term ‘injection-associated increase of protein expression’ refers toa situation, where the total amount of protein that is produced from anRNA administered via jet injection measured for a specific time periodis increased in comparison to the total amount of protein produced fromthe same RNA administered by conventional needle injection or to asituation, where the protein level that is produced from an RNAadministered via jet injection measured for a specific time point isincreased in comparison to the protein level produced from the same RNAadministered by conventional needle injection.

According to the invention, the at least one modification of themodified RNA and the administration by jet injection have a synergisticeffect on the expression of the protein or peptide encoded by themodified RNA. In other terms, the expression increase that is obtainedby using jet injection in order to administer the modified RNA to asubject/tissue compared to the expression that is obtained by usingconventional needle injection in order to administer an unmodifiedreference RNA to a subject/tissue is larger than the sum of theexpression increase obtained by using modified RNA as compared to anunmodified reference RNA, wherein both are administered in the samemanner (“modification-associated increase of protein expression”) andthe expression increase obtained for an (unmodified) RNA administered byjet injection as compared to the same (unmodified) RNA administered bymeans other than jet injection, particularly by conventional needleinjection (“injection-associated increase of protein expression”).

In this context the inventors surprisingly found out that administrationof a modified RNA, which comprises at least one modification whichincreases the protein expression of the encoded protein, by jetinjection increased the expression of the encoded protein much more thanexpected. It was expected that administration by jet injection wouldhave the same effect on protein expression for a modified RNA and for anunmodified reference RNA.

In the context of the present invention, the “modification-associatedprotein expression” may be increased at a specific time point afterinitiation of expression or total protein production over a certain timeperiod after initiation of expression may be increased when compared toan unmodified reference RNA. Thus, the protein level observed at acertain time point after initiation of expression (or after injection,respectively, of the modified RNA), for example, 6, 12, 24, 48, or 72hours post injection, or the total amount of protein produced in a timespan of, e.g. 6, 12, 24, 48 or 72 hours, is preferably higher than theprotein level observed at the same time point after initiation ofexpression (or after injection) or the total protein amount producedwithin the same time span for an unmodified reference RNA.

Preferably, the modification of RNA alone (i.e. without administrationof the modified RNA by jet injection, but using, for instance,conventional needle injection) leads to an expression of the encodedprotein, which is at least 1.5-fold, more preferably at least 2-fold,more preferably at least 5-fold, even more preferably at least 10-fold,most preferably at least 50-fold of the protein production observed fora corresponding, unmodified reference RNA.

The “injection-associated protein expression” may be increased at aspecific time point after initiation of expression (after injection ofthe RNA) or total protein production over a certain time period afterinitiation of expression may be increased when compared to theconventional needle injection of the RNA. Thus, the protein levelobserved at a certain time point after initiation of expression (orafter injection, respectively of the RNA), for example, 6, 12, 24, 48,or 72 hours post injection, or the total amount of protein produced in atime span of, e.g. 6, 12, 24, 48 or 72 hours, is preferably higher thanthe protein level observed at the same time point after initiation ofexpression (or after injection) or the total protein amount producedwithin the same time span for the same RNA injected by a conventionalmethod as for example by needle injection.

Preferably, jet injection increases the protein production from anunmodified reference RNA only to a minor extent e.g. 1.5-fold or 2-fold.

According to the invention, the expression of a protein encoded by anRNA comprising at least one modification and comprising at least oneopen reading frame is further increased (in a synergistic manner) by jetinjection. Thus, protein expression is further increased in comparisonto a reference, such as an RNA having the same modification that isadministered by conventional needle injection. Preferably, the proteinproduction obtained by jet injection of the modified RNA according tothe invention is increased at least 5-fold, more preferably 10-fold,even more preferably 50-fold and even more preferably 100-fold ascompared to an unmodified reference RNA that is administered byconventional means, in particular conventional needle injection.Therefore, the increase in protein production obtained by jet injectionof the modified RNA according to the invention is at least 1.5-fold,more preferably at least 2-fold, more preferably at least 3-fold, morepreferably at least 4-fold, even more preferably at least 5-fold andmost preferably at least 10-fold as compared to the increase in proteinexpression obtained by jet injection of an unmodified reference RNA.Preferably, this holds true for the protein production at a given timepoint post initiation of expression or for total protein production in agiven time period, for example in a time period of 6, 12, 24, 48 or 72hours post initiation of expression or post injection of the RNA.

The term “jet injection”, as used herein, refers to a needle-freeinjection method, wherein a fluid containing at least one modified RNAand, optionally, further suitable excipients is forced through anorifice, thus generating an ultra-fine liquid stream of high pressurethat is capable of penetrating mammalian skin and, depending on theinjection settings, subcutaneous tissue or muscle tissue. In principle,the liquid stream forms a hole in the skin, through which the liquidstream is pushed into the target tissue. Preferably, jet injection isused for intradermal, subcutaneous or intramuscular injection of themodified RNA according to the invention.

The target tissue or rather the layer of tissue, to which the fluid isdelivered by jet injection, depends on several parameters such as thespecific characteristics of the liquid stream that is employed as wellas the physical parameters defining the particular type of skin (andunderlying tissue), to which the fluid is administered. For instance,the density of collagen fibers and the overall elasticity of the tissueto be treated may have an influence on the penetration achieved by theliquid jet. Several physical parameters have an influence on the resultobtained by jet injection. Distinct layers of tissue, which areseparated by mechanical barriers (such as the superficial fascia), canbe specifically addressed by selecting a liquid stream that is suitablefor penetrating deep enough into the skin or underlying tissue withoutpenetrating said mechanical barrier, which is positioned underneath theaddressed layer. E.g., intradermal administration is achieved byapplication of a liquid stream that penetrates into the dermis withoutdisrupting the superficial fascia. The administered fluid distributes inthe dermis horizontally.

Several parameters defining the liquid jet may have an impact on theefficiency of the injection and, in particular, on the depth ofpenetration, which translates into targeting a certain tissue layer,respectively. One such parameter is the pressure, with which the liquidstream hits the skin surface. That pressure is dependent, amongst otherfactors, on the jet exit velocity (i.e. the velocity, at which the jetleaves the nozzle of the injection apparatus) as well as on the distancebetween the nozzle and the skin and the medium (air, liquid) thatconstitutes the space between nozzle and skin. Typically, the velocityof the jet (and the pressure exerted by the jet) is reduced as the jetexits from the nozzle and crosses said space. The penetration of theskin further depends on the jet diameter, which is primarily determinedby the dimensions of the nozzle, which is employed. Notably, comparableresults in terms of tissue penetration and fluid delivery may beobtained by different combinations of parameters.

In order to penetrate the skin, the jet exit velocity of the liquidstream comprising the RNA is preferably at least 60 m/s, more preferablyat least 80 m/s, even more preferably at least 100 m/s and mostpreferably at least 150 m/s.

The liquid stream comprising the RNA that is used in jet injection istypically very fine and is selected such that penetration of the skin isfeasible. Preferably, the liquid jet diameter is selected in accordancewith the desired target tissue. The liquid jet diameter is preferablyregulated by the nozzle orifice, i.e. the liquid jet diameter increaseswith increasing diameter of the nozzle orifice. Preferably, the liquidjet diameter corresponds to the orifice diameter so that the liquid jetdiameter is equal or slightly larger than the diameter of the nozzleorifice. Typically, at constant jet exit velocity, the penetration depthachieved in the tissue will be higher for greater liquid jet diameters.

In one embodiment of the invention, the diameter of the orifice isbetween 20 μm and 600 μm, preferably between 100 μm and 300 μm, morepreferably between 120 μm and 250 m.

In a further preferred embodiment, the diameter of the orifice isbetween 20 μm and 150 am, preferably between 30 μm and 130 μm, morepreferably between 40 μm and 110 μm, even more preferably between 50 μmand 100 m.

In another embodiment, the diameter of the orifice is between 70 μm and300 μm, preferably between 80 μm and 200 μm, more preferably between 90μm and 180 μm, even more preferably between 100 μm and 150 μm.

Preferably, the injection time (i.e. the time between the first contactof the jet with the target skin and the time point of jet cessation) isless than 1.0 seconds, more preferably less than 0.7 seconds and evenmore preferably less than 0.3 seconds. Most preferably the injectiontime is less than 0.1 second.

In a preferred embodiment, the process of jet injection comprises atleast two phases characterized by different jet velocities. Preferably,jet injection begins with a first phase, wherein a first jet velocity isselected so as to ensure tissue penetration. Said first jet velocity isdependent to a large extent on the exit jet velocity, i.e. the velocity,at which the liquid jet leaves the device's nozzle. Said first velocityis further adapted to the desired injection depth. Subsequently, asecond jet velocity is employed in a second phase, which is appropriateto deliver the fluid into the target tissue. Said second jet velocity istypically lower than the first velocity and is chosen as to not exceedthe absorption capacity of the tissue.

In a preferred embodiment, the jet injection of the modified RNAaccording to the invention comprises three phases:

The initial penetration phase is characterized by the highest pressurewith respect to the pressure profile of the whole jet injection process(therefore also referred to as peak pressure phase). The penetrationphase preferably lasts less than 50 ms, more preferably less than 10 ms,even more preferably less than 5 ms. Most preferably the penetrationphase lasts less than 1 ms.

After the peak pressure phase, the pressure is reduced in the deliveryphase, while maintaining a level sufficient for injecting the liquidstream into the target tissue. It is preferred that the pressure levelis constant or decreases only slowly during the delivery phase.Preferably, the delivery phase lasts less than 0.8 seconds, morepreferably less than 0.5 seconds, even more preferably the deliveryphase lasts from 0.01 to 0.3 seconds, most preferably from 0.01 to 0.1seconds.

The final stage of the jet injection process according to thisembodiment of the invention is characterized by a drop of the pressureacting on the liquid comprising the modified RNA to levels around theatmospheric pressure level (drop-off phase). Typically, the pressuredrops abruptly after the delivery phase. The drop-off phase preferablylasts less than 0.3 seconds, more preferably less than 0.2 seconds, evenmore preferably less than 50 milliseconds, most preferably less than 10milliseconds.

Depending on the subject to be treated, the target tissue and thespecific application, the volume of the liquid comprising the modifiedRNA is selected accordingly. In a preferred embodiment of the invention,the volume of the administered liquid is between 0.05 μl and 1000 μl,preferably between 0.1 μl and 500 μl, more preferably between 0.2 μl and200 μl. Most preferably the volume of the liquid comprising the modifiedRNA is 100 μl.

In this context, a volume of the liquid comprising the modified RNA ispreferably between 0.2 and 200 μl, and is most preferably 100 μl if themodified RNA is injected intradermally by jet injection.

Furthermore, the volume of the liquid comprising the modified RNA ispreferably between 100 and 2000 μl and is most preferably 500 μl, if themodified RNA is injected intramuscularly by jet injection.

If the liquid comprising the modified RNA is injected subcutaneously byjet injection, the volume of the liquid comprising the modified RNA ispreferably between 100 and 2000 μl and is most preferably 500 μl.

In the meaning of the present invention, any device may be used for jetinjection as long as it is capable of generating a liquid jet that issuitable for delivery as defined herein. There is no limitation as tothe means, by which the liquid is accelerated. For instance, systemsusing springs to expell the liquid may be employed as well as systemsusing gas or other propellants. Furthermore, a constant liquid jet maybe used, preferably with at least two distinct velocities in at leasttwo phases as described herein. Alternatively, a pulsed microjet may beused. Preferably, jet injection systems are used that are commerciallyavailable, such as Stratis, Tropis (both from Pharmajet), Vitajet,Biojector 2000 or Bioject Zetajet (all three from Bioject MedicalTechnologies Inc.), Glide (from Glide Pharma), MediJector Vision (fromAntares), Sumavel DosePro (from Zogenix), SQ Pen (from Bespak), andInjex (from Equidyne). The modified RNA is injected by using a system,which preferably allows precise and reproducible delivery of apreselected dosage. Preferably, the device ensures suitable tensioningof the skin in order for the liquid jet to be injected into the skin. Ina preferred embodiment, a device as disclosed in the internationalpatent application WO 2013/090315, the disclosure of which is includedherein in its entirety. Preferably, the modified RNA is injected byusing a device that comprises at least one of the individual componentsof the needle-free injection device as shown in any of FIGS. 1 to 17C ofWO 2013/090315, preferably a component selected from the groupconsisting of a compressible main spring, an actuation button, a skintensioning spring, a plunger body, a seal and a hammer. More preferably,the modified RNA is injected using a device as disclosed in any of FIGS.1 to 10 of WO 2013/090315, wherein the main spring can preferably becaused to move laterally away from a syringe end of the injection devicewhen pressure is applied to the needle-free syringe nozzle prior to aninjection. In a particularly preferred embodiment, the modified RNA isinjected by using a Tropis device (Pharmajet).

According to the invention, an RNA comprising at least one modificationand comprising at least one open reading frame is administered by jetinjection intradermally, intramuscularly or subcutaneously. In a furtherpreferred embodiment, the modified RNA is administered intradermally,wherein jet injection is performed by using a nozzle that has a diameterof between 20 μm and 150 μm, preferably of between 30 μm and 130 μm,more preferably of between 40 μm and 110 am, even more preferably ofbetween 50 μm and 100 μm and the jet exit velocity is preferably atleast 80 m/s, more preferably at least 100 m/s, even more preferably atleast 150 m/s and most preferably at least 190 m/s.

In a preferred embodiment, the modified RNA is administeredintradermally by using the TROPIS device (Pharmajet).

Typically, the successful delivery of a liquid to the dermis of amammalian can be assessed by the formation of a wheal (also referred toas bleb) at the site of injection. Preferably, the wheal diameter is atleast 5 mm, more preferably at least 7 mm, even more preferably at least9 and most preferably at least 10 mm.

In the meaning of the present invention, the term “modified RNA”comprises any type of ribonucleic acid molecule that is modified suchthat the amount of protein or peptide produced from said modified RNAmeasured at a specific time point or over a specific time period isincreased in comparison with RNA lacking the modification (“unmodifiedreference RNA”).

This increase in expression is termed herein as “modification-associatedincrease of protein expression” as defined above.

According to the invention, a modified RNA molecule is provided, whichis characterized by increased expression of the encoded protein incomparison to a respective RNA lacking the modification (“unmodifiedreference RNA”). In order to assess the in vivo protein production by amodified RNA, the expression of the encoded protein is determinedfollowing injection of the modified RNA into target cells/tissue andcompared to the protein expression of the unmodified reference RNA.Quantitative methods for determining protein expression are known in theart (e.g. Western-Blot, ELISA, mass spectometry). Particularly useful inthis context is the determination of the expression of reporter proteinslike luciferase or secreted alkaline phosphatase (SEAP). Thus, modifiedRNA or unmodified reference RNA is injected into the target tissue, e.g.via conventional needle injection or via jet injection, preferablyintradermally, intramuscularly or subcutanously. Several hours orseveral days (e.g. 6, 12, 24, 48 or 72 hours) post initiation ofexpression or post injection of the RNA, tissue from the injection siteis collected and lysed. Afterwards the lysates can be used to detect andquantify the expressed protein (and thus determine the efficiency ofprotein expression) using several methods, e.g. Western-Blot, ELISA,mass spectrometry or by fluorescence or luminescence measurement.

Therefore, if the protein expression from a modified RNA is compared tothe protein expression from an unmodified reference RNA at a specifictime point (e.g. 6, 12, 24, 48 or 72 hours post initiation of expressionor post injection of the RNA), both RNAs are injected separately intotest animals, tissue from the injection sites is collected after aspecific time point, protein lysates are prepared according to theparticular protocol adjusted to the particular detection method (e.g.Western Blot, ELISA, etc. as known in the art) and the protein isdetected by the chosen detection method and quantified.

If the total amount of protein for a specific time period is to bemeasured, tissue can be collected after several time points afterinjection of the RNA (e.g. 6, 12, 24, 48 and 72 hours post initiation ofexpression or post injection of the RNA; usually from different testanimals), and the protein amount per time point can be determined asexplained above. In order to calculate the cumulative protein amount, amathematical method of determining the total amount of protein can beused, e.g. the area under the curve (AUC) can be determined according tothe following formula:

${AUC} = {\int\limits_{a}^{b}{{f(x)}\mspace{11mu} {d(x)}}}$

In order to calculate the area under the curve for the total amount ofprotein, the integral of the equation of the expression curve from eachend point (a and b) is calculated.

According to the invention, an RNA molecule is structurally modified inorder to enhance the expression of the encoded protein(“modification-associated increase of protein expression”). Therein, themodification is not limited to any particular structure as long as theexpression of the encoded protein is increased as compared to anunmodified reference RNA as defined herein. Several RNA modificationsare known in the art, which can be applied to a given RNA in the contextof the present invention.

Chemical Modifications:

The term “RNA modification” as used herein may refer to chemicalmodifications comprising backbone modifications as well as sugarmodifications or base modifications.

In this context, the modified RNA molecule as defined herein may containnucleotide analogues/modifications, e.g. backbone modifications, sugarmodifications or base modifications. A backbone modification inconnection with the present invention is a modification, in whichphosphates of the backbone of the nucleotides contained in a nucleicacid molecule as defined herein are chemically modified. A sugarmodification in connection with the present invention is a chemicalmodification of the sugar of the nucleotides of the nucleic acidmolecule as defined herein. Furthermore, a base modification inconnection with the present invention is a chemical modification of thebase moiety of the nucleotides of the nucleic acid molecule of thenucleic acid molecule. In this context nucleotide analogues ormodifications are preferably selected from nucleotide analogues whichare applicable for transcription and/or translation.

Sugar Modifications:

The modified nucleosides and nucleotides, which may be incorporated intothe modified RNA as described herein, can be modified in the sugarmoiety. For example, the 2′ hydroxyl group (OH) can be modified orreplaced with a number of different “oxy” or “deoxy” substituents.Examples of “oxy”-2′ hydroxyl group modifications include, but are notlimited to, alkoxy or aryloxy (—OR, e.g., R═H, alkyl, cycloalkyl, aryl,aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),-0(CH2CH2o)nCH2CH2OR; “locked” nucleic acids (LNA) in which the 2′hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon ofthe same ribose sugar; and amino groups (—O-amino, wherein the aminogroup, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl,arylamino, diarylamino, heteroarylamino, or diheteroaryl amino, ethylenediamine, polyamino) or aminoalkoxy.

“Deoxy” modifications include hydrogen, amino (e.g. NH2; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,diheteroaryl amino, or amino acid); or the amino group can be attachedto the sugar through a linker, wherein the linker comprises one or moreof the atoms C, N, and O.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA can include nucleotidescontaining, for instance, arabinose as the sugar.

Backbone Modifications:

The phosphate backbone may further be modified in the modifiednucleosides and nucleotides, which may be incorporated into the modifiedRNA, as described herein. The phosphate groups of the backbone can bemodified by replacing one or more of the oxygen atoms with a differentsubstituent. Further, the modified nucleosides and nucleotides caninclude the full replacement of an unmodified phosphate moiety with amodified phosphate as described herein. Examples of modified phosphategroups include, but are not limited to, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. The phosphate linker can also be modified by thereplacement of a linking oxygen with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylene-phosphonates).

Base Modifications:

The modified nucleosides and nucleotides, which may be incorporated intothe modified RNA, as described herein, can further be modified in thenucleobase moiety. Examples of nucleobases found in RNA include, but arenot limited to, adenine, guanine, cytosine and uracil. For example, thenucleosides and nucleotides described herein can be chemically modifiedon the major groove face. In some embodiments, the major groove chemicalmodifications can include an amino group, a thiol group, an alkyl group,or a halo group.

In particularly preferred embodiments of the present invention, thenucleotide analogues/modifications are selected from base modifications,which are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-O-Methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5′-triphosphate,5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-Iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-Iodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5′-triphosphate,5-Propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladeno sine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleotide can be modified on the major grooveface and can include replacing hydrogen on C-5 of uracil with a methylgroup or a halo group.

In specific embodiments, a modified nucleoside is5′-O-(l-Thiophosphate)-Adenosine, 5′-O-(1-Thiophosphate)-Cytidine,5′-O-(1-Thiophosphate)-Guanosine, 5′-O-(1-Thiophosphate)-Uridine or5′-O-(l-Thiophosphate)-Pseudouridine.

In further specific embodiments the modified RNA may comprise nucleosidemodifications selected from 6-aza-cytidine, 2-thio-cytidine,α-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine,5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine,α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine,deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine,α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine,7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-Chloro-purine,N6-methyl-2-amino-purine, Pseudo-iso-cytidine, 6-Chloro-purine,N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine,7-deaza-adenosine.

Lipid Modification:

According to a further embodiment, the modified RNA as defined hereincan contain a lipid modification. Such a lipid-modified RNA typicallycomprises an RNA as defined herein. Such a lipid-modified RNA moleculeas defined herein typically further comprises at least one linkercovalently linked with that RNA molecule, and at least one lipidcovalently linked with the respective linker. Alternatively, thelipid-modified RNA molecule comprises at least one RNAmolecule asdefined herein and at least one (bifunctional) lipid covalently linked(without a linker) with that RNA molecule. According to a thirdalternative, the lipid-modified RNA molecule comprises an RNA moleculeas defined herein, at least one linker covalently linked with that RNAmolecule, and at least one lipid covalently linked with the respectivelinker, and also at least one (bifunctional) lipid covalently linked(without a linker) with that RNA molecule. In this context, it isparticularly preferred that the lipid modification is present at theterminal ends of a linear RNA sequence.

Modification of the 5′-End of the Modified RNA:

According to another preferred embodiment of the invention, the modifiedRNA molecule as defined herein, can be modified by the addition of aso-called “5′ CAP” structure. A 5′-cap is an entity, typically amodified nucleotide entity, which generally “caps” the 5′-end of amature mRNA. A 5′-cap may typically be formed by a modified nucleotide,particularly by a derivative of a guanine nucleotide. Preferably, the5′-cap is linked to the 5′-terminus via a 5′-5′-triphosphate linkage. A5′-cap may be methylated, e.g. m7GpppN, wherein N is the terminal 5′nucleotide of the nucleic acid carrying the 5′-cap, typically the 5′-endof an RNA. m7GpppN is the 5′-CAP structure which naturally occurs inmRNA transcribed by polymerase II and is therefore not considered asmodification comprised in the modified RNA according to the invention.This means the modified RNA according to the present invention maycomprise a m7GpppN as 5′-CAP, but additionally the modified RNAcomprises at least one further modification as defined herein.

Further examples of 5′cap structures include glyceryl, inverted deoxyabasic residue (moiety), 4′,5′ methylene nucleotide,1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides,alpha-nucleotide, modified base nucleotide, threo-pentofuranosylnucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutylnucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-invertednucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-invertednucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediolphosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate,3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging ornon-bridging methylphosphonate moiety. These modified 5′-CAP structuresare regarded as at least one modification comprised in the modified RNAaccording to the present invention.

Particularly preferred modified 5′-CAP structures are CAP1 (methylationof the ribose of the adjacent nucleotide of m7G), CAP2 (methylation ofthe ribose of the 2^(nd) nucleotide downstream of the m7G), CAP3(methylation of the ribose of the 3^(rd) nucleotide downstream of them7G), CAP4 (methylation of the ribose of the 4^(th) nucleotidedownstream of the m7G), ARCA (anti-reverse CAP analogue, modified ARCA(e.g. phosphothioate modified ARCA), inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

Sequence Modification of the Open Reading Frame: Modification of the G/CContent:

In a particularly preferred embodiment of the present invention, the G/Ccontent of the coding region, encoding at least one peptide or proteinof the modified RNA as defined herein, is modified, particularlyincreased, compared to the G/C content of its particular wild typecoding region, i.e. the unmodified coding region. The encoded amino acidsequence of the coding region is preferably not modified compared to thecoded amino acid sequence of the particular wild type coding region.

The modification of the G/C-content of the coding region of the modifiedRNA as defined herein is based on the fact that the sequence of any mRNAregion to be translated is important for efficient translation of thatmRNA. Thus, the composition and the sequence of various nucleotides areimportant. In particular, mRNA sequences having an increased G(guanosine)/C (cytosine) content are more stable than mRNA sequenceshaving an increased A (adenosine)/U (uracil) content. According to theinvention, the codons of the coding region are therefore varied comparedto its wild type coding region, while retaining the translated aminoacid sequence, such that they include an increased amount of G/Cnucleotides. In respect to the fact that several codons code for one andthe same amino acid (so-called degeneration of the genetic code), themost favourable codons for the stability can be determined (so-calledalternative codon usage).

Depending on the amino acid to be encoded by the coding region of themodified RNA as defined herein, there are various possibilities formodification of the RNA sequence, e.g. the coding region, compared toits wild type coding region. In the case of amino acids, which areencoded by codons, which contain exclusively G or C nucleotides, nomodification of the codon is necessary. Thus, the codons for Pro (CCC orCCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) require nomodification, since no A or U is present.

In contrast, codons which contain A and/or U nucleotides can be modifiedby substitution of other codons which code for the same amino acids butcontain no A and/or U. Examples of these are:

the codons for Pro can be modified from CCU or CCA to CCC or CCG;the codons for Arg can be modified from CGU or CGA or AGA or AGG to CGCor CGG;the codons for Ala can be modified from GCU or GCA to GCC or GCG;the codons for Gly can be modified from GGU or GGA to GGC or GGG.

In other cases, although A or U nucleotides cannot be eliminated fromthe codons, it is however possible to decrease the A and U content byusing codons, which contain a lower content of A and/or U nucleotides.Examples of these are:

the codons for Phe can be modified from UUU to UUC;the codons for Leu can be modified from UUA, UUG, CUU or CUA to CUC orCUG;the codons for Ser can be modified from UCU or UCA or AGU to UCC, UCG orAGC;the codon for Tyr can be modified from UAU to UAC;the codon for Cys can be modified from UGU to UGC;the codon for His can be modified from CAU to CAC;the codon for Gln can be modified from CAA to CAG;the codons for Ile can be modified from AUU or AUA to AUC;the codons for Thr can be modified from ACU or ACA to ACC or ACG;the codon for Asn can be modified from AAU to AAC;the codon for Lys can be modified from AAA to AAG;the codons for Val can be modified from GUU or GUA to GUC or GUG;the codon for Asp can be modified from GAU to GAC;the codon for Glu can be modified from GAA to GAG;the stop codon UAA can be modified to UAG or UGA.

In the case of the codons for Met (AUG) and Trp (UGG), on the otherhand, there is no possibility of sequence modification.

The substitutions listed above can be used either individually or in anypossible combination to increase the G/C content of the coding region ofthe modified RNA as defined herein, compared to its particular wild typecoding region (i.e. the original sequence). Thus, for example, allcodons for Thr occurring in the wild type sequence can be modified toACC (or ACG).

Preferably, the G/C content of the coding region of the modified RNA asdefined herein is increased by at least 7%, more preferably by at least15%, particularly preferably by at least 20%, compared to the G/Ccontent of the wild type coding region. According to a specificembodiment at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably atleast 70%, even more preferably at least 80% and most preferably atleast 90%, 95% or even 100% of the substitutable codons in the codingregion encoding at least one peptide or protein, which comprises apathogenic antigen or a fragment, variant or derivative thereof, aresubstituted, thereby increasing the G/C content of said coding region.

In this context, it is particularly preferable to increase the G/Ccontent of the coding region of the modified RNA as defined herein, tothe maximum (i.e. 100% of the substitutable codons), compared to thewild type coding region.

Codon Optimization:

According to the invention, a further preferred modification of thecoding region encoding at least one peptide or protein of the modifiedRNA as defined herein, is based on the finding that the translationefficiency is also determined by a different frequency in the occurrenceof tRNAs in cells. Thus, if so-called “rare codons” are present in thecoding region of the wild type RNA sequence, to an increased extent, themRNA is translated to a significantly poorer degree than in the casewhere codons coding for relatively “frequent” tRNAs are present.

In this context, the coding region of the modified RNA is preferablymodified compared to the corresponding wild type coding region such thatat least one codon of the wild type sequence, which codes for a tRNAwhich is relatively rare in the cell, is exchanged for a codon, whichcodes for a tRNA which is relatively frequent in the cell and carriesthe same amino acid as the relatively rare tRNA. By this modification,the coding region of the modified RNA as defined herein, is modifiedsuch that codons, for which frequently occurring tRNAs are available,are inserted. In other words, according to the invention, by thismodification all codons of the wild type coding region, which code for atRNA which is relatively rare in the cell, can in each case be exchangedfor a codon, which codes for a tRNA which is relatively frequent in thecell and which, in each case, carries the same amino acid as therelatively rare tRNA.

Which tRNAs occur relatively frequently in the cell and which, incontrast, occur relatively rarely is known to a person skilled in theart; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. Thecodons which use for the particular amino acid the tRNA which occurs themost frequently, e.g. the Gly codon, which uses the tRNA which occursthe most frequently in the (human) cell, are particularly preferred.

According to the invention, it is particularly preferable to link thesequential G/C content, which is increased, in particular maximized, inthe coding region of the modified RNA as defined herein, with the“frequent” codons without modifying the amino acid sequence of thepeptide or protein encoded by the coding region of the RNA sequence.This preferred embodiment allows provision of a particularly efficientlytranslated and stabilized (modified) RNA sequence as defined herein.

Modification of the 3′-End of the Modified RNA:

Furthermore, in the modified RNA comprising at least one open readingframe that is to be administered by jet injection, the region 3′ of thecoding region may be modified, e.g. by insertion or addition ofstretches of multiple repeats of adenine or cytosine residues. In apreferred embodiment of the invention, the 3′ end of an RNA molecule ismodified by addition of a series of adenine nucleotides (“poly(A)tail”).

The length of the poly(A) tail has a major impact on translationefficiency of the respective construct. To be translated efficiently,the poly(A) tail of exogenously delivered mRNAs should consist of atleast 20 A residues. Moreover, it has been described that mRNAexpression positively correlates with poly(A) tail length (reviewed inTavernier et al., J Control Release. 2011 Mar. 30; 150(3):238-47. PMID:20970469). In this context Holtkamp et al. showed that a poly(A) tailmeasuring 120 nucleotides compared with a shorter one, enhanced mRNAstability and translational efficiency (Holtkamp et al., Blood. 2006Dec. 15; 108(13):4009-17. PMID: 16940422). In one embodiment of theinvention, modified RNA is used for jet injection, which comprises(optionally in combination with other modifications) a poly(A) tail,wherein the poly(A) tail comprises at least 30, preferably more than 50,more preferably more than 100, even more preferably more than 200adenine nucleotides. Most preferably the RNA comprises a poly(A) tailconsisting of 64 adenine nucleotides.

In specific embodiments a poly(A) tail is only considered as at leastone modification comprised in the modified RNA according to the presentinvention if the poly(A) tail is longer than 30 adenosines, preferablylonger than 50 adenosines, more preferably longer than 100 adenosinesand even more preferably longer than 200 adenosines.

In further specific embodiments, the modification with a poly(A) tail isnot considered as at least one modification of the modified RNAaccording to the present invention. This means that the modified RNAaccording to the invention may comprise a poly(A) tail as defined abovebut it additionally comprises at least one further modification asdefined herein.

In a preferred embodiment, the modified RNA comprising at least one openreading frame which is used for jet injection, comprises (optionally incombination with other modifications) a poly(C) sequence in the region3′ of the coding region of the RNA. A poly(C) sequence is typically astretch of multiple cytosine nucleotides, typically about 10 to about200 cytidine nucleotides, preferably about 10 to about 100 cytidinenucleotides, more preferably about 10 to about 70 cytidine nucleotidesor even more preferably about 20 to about 50 or even about 20 to about30 cytidine nucleotides. A poly(C) sequence may preferably be located 3′of the coding region comprised by a nucleic acid. In a specificpreferred embodiment of the present invention, the poly(C) sequence islocated 3′ of a poly(A) sequence.

In a further preferred embodiment, the modified RNA according to theinvention comprises or codes for a histone stem-loop at its 3′ terminusas at least one modification. In the context of the present invention, ahistone stem-loop sequence is preferably selected from at least one ofthe following formulae (I) or (II):

formula (I) (stem-loop sequence without stem bordering elements):

formula (II) (stem-loop sequence with stem bordering elements):

wherein:

stem1 or stem2 is a consecutive sequence of 1 to 6, preferably of 2 to6, bordering more preferably of 2 to 5, even more preferably of 3 toelements N₁₋₆ 5, most preferably of 4 to 5 or 5 N, wherein each N isindependently from another selected from a nucleotide selected from A,U, T, G and C, or a nucleotide analogue thereof; stem1 is reversecomplementary or partially reverse [N₀₋₂GN₃₋₅] complementary withelement stem2, and is a consecutive sequence between of 5 to 7nucleotides; wherein N₀₋₂ is a consecutive sequence of 0 to 2,preferably of 0 to 1, more preferably of 1 N, wherein each N isindependently from another selected from a nucleotide selected from A,U, T, G and C or anucleotide analogue thereof; wherein N₃₋₅ is aconsecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of4 N, wherein each N is independently from another selected from anucleotide selected from A, U, T, G and C or a nucleotide analoguethereof, and wherein G is guanosine or an analogue thereof, and may beoptionally replaced by a cytidine or an analogue thereof, provided thatits complementary nucleotide cytidine in stem2 is replaced by guanosine;loop sequence is located between elements stem1 and stem2, and is[N₀₋₄(U/T)N₀₋₄] a consecutive sequence of 3 to 5 nucleotides, morepreferably of 4 nucleotides; wherein each N₀₋₄ is independent fromanother a consecutive sequence of 0 to 4, preferably of 1 to 3, morepreferably of 1 to 2 N, wherein each N is independently from anotherselected from a nucleotide selected from A, U, T, G and C or anucleotide analogue thereof; and wherein U/T represents uridine, oroptionally thymidine; stem2 is reverse complementary or partiallyreverse [N₃₋₅CN₀₋₂] complementary with element stem1, and is aconsecutive sequence between of 5 to 7 nucleotides; wherein N₃₋₅ is aconsecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of4 N, wherein each N is independently from another selected from anucleotide selected from A, U, T, G and C or a nucleotide analoguethereof; wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of0 to 1, more preferably of 1 N, wherein each N is independently fromanother selected from a nucleotide selected from A, U, T, G or C or anucleotide analogue thereof; and wherein C is cytidine or an analoguethereof, and may be optionally replaced by a guanosine or an analoguethereof provided that its complementary nucleoside guanosine in stem1 isreplaced by cytidine;whereinstem1 and stem2 are capable of base pairing with each other forming areverse complementary sequence, wherein base pairing may occur betweenstem1 and stem2, e.g. by Watson-Crick base pairing of nucleotides A andU/T or G and C or by non-Watson-Crick base pairing e.g. wobble basepairing, reverse Watson-Crick base pairing, Hoogsteen base pairing,reverse Hoogsteen base pairing or are capable of base pairing with eachother forming a partially reverse complementary sequence, wherein anincomplete base pairing may occur between stem1 and stem2, on the basisthat one or more bases in one stem do not have a complementary base inthe reverse complementary sequence of the other stem.

According to a further preferred embodiment, the modified RNA accordingto the present invention may comprise or code for at least one histonestem-loop sequence according to at least one of the following specificformulae (Ia) or (IIa):

formula (Ia) (stem-loop sequence without stem bordering elements):

formula (IIa) (stem-loop sequence with stem bordering elements):

wherein:N, C, G, T and U are as defined above.

According to a further more particularly preferred embodiment, themodified RNA may comprise or code for at least one histone stem-loopsequence according to at least one of the following specific formulae(Ib) or (IIb):

formula (Ib) (stem-loop sequence without stem bordering elements):

formula (IIb) (stem-loop sequence with stem bordering elements):

wherein: N, C, G, T and U are as defined above.

According to an even more preferred embodiment, the modified RNAaccording to the present invention may comprise or code for at least onehistone stem-loop sequence according to at least one of the followingspecific formulae (Ic) to (Ih) or (IIc) to (IIh), shown alternatively inits stem-loop structure and as a linear sequence representing histonestem-loop sequences as generated according to

Example 1

formula (Ic): (metazoan and protozoan histone stem-loop consensussequence without stem bordering elements):

(SEQ ID NO: 1)      N U     N   N      N-N      N-N      N-N      N-N     G-C      N-N (stem-loop structure) NGNNNNNNUNNNNNCN (linearsequence)formula (IIc): (metazoan and protozoan histone stem-loop consensussequence with stem bordering elements):

(SEQ ID NO: 2)       N U      N   N       N-N       N-N       N-N      N-N       G-C N*N*NNNN-NNNN*N*N* (stem-loop structure)N*N*NNNNGNNNNNNUNNNNNCNNNN*N*N* (linear sequence)formula (Id): (without stem bordering elements)

(SEQ ID NO: 3)      N U     N   N      N-N      N-N      N-N      N-N     C-G      N-N (stem-loop structure) NCNNNNNNUNNNNNGN (linearsequence)formula (KKd): (with stem bordering elements)

(SEQ ID NO: 4)        N U       N   N        N-N        N-N        N-N       N-N        C-G N*N*NNNN-NNNN*N*N* (stem-loop structure)N*N*NNNNCNNNNNNUNNNNNGNNNN*N*N* (linear sequence)formula (Ie): (protozoan histone stem-loop consensus sequence withoutstem bordering elements)

(SEQ ID NO: 5)      N U     N   N      N-N      N-N      N-N      N-N     G-C      D-H (stem-loop structure) DGNNNNNNUNNNNNCH (linearsequence)formula (IIe): (protozoan histone stem-loop consensus sequence with stembordering elements)

(SEQ ID NO: 6)        N U       N   N        N-N        N-N        N-N       N-N        G-C N*N*NNND-HNNN*N*N* (stem-loop structure)N*N*NNNDGNNNNNNUNNNNNCHNNN*N*N* (linear sequence)formula (If): (metazoan histone stem-loop consensus sequence withoutstem bordering elements)

(SEQ ID NO: 7)      N U     N    N      Y-V      Y-N      B-D      N-N     G-C      N-N (stem-loop structure) NGNBYYNNUNVNDNCN (linearsequence)formula (IIf): (metazoan histone stem-loop consensus sequence with stembordering elements)

(SEQ ID NO: 8)        N U       N    N        Y-V        Y-N        B-D       N-N        G-C N*N*NNNN-NNNN*N*N* (stem-loop structure)N*N*NNNNGNBYYNNUNVNDNCNNNN*N*N* (linear sequence)formula (Ig): (vertebrate histone stem-loop consensus sequence withoutstem bordering elements)

(SEQ ID NO: 9)      N U     D   H      Y-A      Y-B      Y-R      H-D     G-C      N-N (stem-loop structure) NGHYYYDNUHABRDCN (linearsequence)formula (IIg): (vertebrate histone stem-loop consensus sequence withstem bordering elements)

(SEQ ID NO: 10)        N U       D   H        Y-A        Y-B        Y-R       H-D        G-C N*N*HNNN-NNNN*N*H* (stem-loop structure)N*N*HNNNGHYYYDNUHABRDCNNNN*N*H* (linear sequence)formula (Ih): (human histone stem-loop consensus sequence (Homo sapiens)without stem bordering elements)

(SEQ ID NO: 11)      Y U     D   H      U-A      C-S      Y-R      H-R     G-C      D-C (stem-loop structure) DGHYCUDYUHASRRCC (linearsequence)formula (IIh): (human histone stem-loop consensus sequence (Homosapiens) with stem bordering elements)

(SEQ ID NO: 12)        Y U       D   H        U-A        C-S        Y-R       H-R        G-C N*H*AAHD-CVHB*N*H* (stem loop structure)N*H*AAHDGHYCUDYUHASRRCCVHB*N*H* (linear sequence)wherein in each of above formulae (Ic) to (Ih) or (IIc) to (IIh): N, C,G, A, T and U are as defined above; each U may be replaced by T; each(highly) conserved G or C in the stem elements 1 and 2 may be replacedby its complementary nucleotide base C or G, provided that itscomplementary nucleotide in the corresponding stem is replaced by itscomplementary nucleotide in parallel; and/or G, A, T, U, C, R, Y, M, K,S, W, H, B, V, D, and N are nucleotide bases as defined in the followingTable:

abbreviation Nucleotide bases remark G G Guanine A A Adenine T T ThymineU U Uracile C C Cytosine R G or A Purine Y T/U or C Pyrimidine M A or CAmino K G or T/U Keto S G or C Strong (3H bonds) W A or T/U Weak (2Hbonds) H A or C or T/U Not G B G or T/U or C Not A V G or C or A Not T/UD G or A or T/U Not C N G or C or T/U or A Any base * Present or notBase may be present or not

In this context, it is particularly preferred that the histone stem-loopsequence according to at least one of the formulae (I) or (Ia) to (Ih)or (II) or (IIa) to (IIh) of the present invention is selected from anaturally occurring histone stem loop sequence, more particularlypreferred from protozoan or metazoan histone stem-loop sequences, andeven more particularly preferred from vertebrate and mostly preferredfrom mammalian histone stem-loop sequences especially from human histonestem-loop sequences.

According to a particularly preferred embodiment, the histone stem-loopsequence according to at least one of the specific formulae (I) or (Ia)to (Ih) or (II) or (IIa) to (IIh) of the present invention is a histonestem-loop sequence comprising at each nucleotide position the mostfrequently occurring nucleotide, or either the most frequently or thesecond-most frequently occurring nucleotide of naturally occurringhistone stem-loop sequences in metazoa and protozoa, protozoa, metazoa,vertebrates and humans. In this context it is particularly preferredthat at least 80%, preferably at least 85%, or most preferably at least90% of all nucleotides correspond to the most frequently occurringnucleotide of naturally occurring histone stem-loop sequences.

In a further particular embodiment, the histone stem-loop sequenceaccording to at least one of the specific formulae (I) or (Ia) to (Ih)of the present invention is selected from following histone stem-loopsequences (without stem-bordering elements):

(SEQ ID NO: 13 according to formula (Ic)) VGYYYYHHTHRVVRCB (SEQ ID NO:14 according to formula (Ic)) SGYYYTTYTMARRRCS (SEQ ID NO: 15 accordingto formula (Ic)) SGYYCTTTTMAGRRCS (SEQ ID NO: 16 according to formula(Ie)) DGNNNBNNTHVNNNCH (SEQ ID NO: 17 according to formula (Ie))RGNNNYHBTHRDNNCY (SEQ ID NO: 18 according to formula (Ie))RGNDBYHYTHRDHNCY (SEQ ID NO: 19 according to formula (If))VGYYYTYHTHRVRRCB (SEQ ID NO: 20 according to formula (If))SGYYCTTYTMAGRRCS (SEQ ID NO: 21 according to formula (If))SGYYCTTTTMAGRRCS (SEQ ID NO: 22 according to formula (Ig))GGYYCTTYTHAGRRCC (SEQ ID NO: 23 according to formula (Ig))GGCYCTTYTMAGRGCC (SEQ ID NO: 24 according to formula (Ig))GGCTCTTTTMAGRGCC (SEQ ID NO: 25 according to formula (Ih))DGHYCTDYTHASRRCC (SEQ ID NO: 26 according to formula (Ih))GGCYCTTTTHAGRGCC (SEQ ID NO: 27 according to formula (Ih))GGCYCTTTTMAGRGCC

Furthermore in this context, following histone stem-loop sequences (withstem bordering elements) according to one of specific formulae (II) or(IIa) to (IIh) are particularly preferred:

(SEQ ID NO: 28 according to formula (IIc))H*H*HHVVGYYYYHHTHRVVRCBVHH*N*N* (SEQ ID NO: 29 according to formula(IIc)) M*H*MHMSGYYYTTYTMARRRCSMCH*H*H* (SEQ ID NO: 30 according toformula (IIc)) M*M*MMMSGYYCTTTTMAGRRCSACH*M*H* (SEQ ID NO: 31 accordingto formula (IIe)) N*N*NNNDGNNNBNNTHVNNNCHNHN*N*N* (SEQ ID NO: 32according to formula (IIe)) N*N*HHNRGNNNYHBTHRDNNCYDHH*N*N* (SEQ ID NO:33 according to formula (IIe)) N*H*HHVRGNDBYHYTHRDHNCYRHH*H*H* (SEQ IDNO: 34 according to formula (IIf)) H*H*MHMVGYYYTYHTHRVRRCBVMH*H*N* (SEQID NO: 35 according to formula (IIf)) M*M*MMMSGYYCTTYTMAGRRCSMCH*H*H*(SEQ ID NO: 36 according to formula (IIf))M*M*MMMSGYYCTTTTMAGRRCSACH*M*H* (SEQ ID NO: 37 according to formula(IIg)) H*H*MAMGGYYCTTYTHAGRRCCVHN*N*M* (SEQ ID NO: 38 according toformula (IIg)) H*H*AAMGGCYCTTYTMAGRGCCVCH*H*M* (SEQ ID NO: 39 accordingto formula (IIg)) M*M*AAMGGCTCTTTTMAGRGCCMCY*M*M* (SEQ ID NO: 40according to formula (IIh)) N*H*AAHDGHYCTDYTHASRRCCVHB*N*H* (SEQ ID NO:41 according to formula (IIh)) H*H*AAMGGCYCTTTTHAGRGCCVMY*N*M* (SEQ IDNO: 42 according to formula (IIh)) H*M*AAAGGCYCTTTTMAGRGCCRMY*H*M*

According to a further preferred embodiment, the modified RNA comprisesor codes for at least one histone stem-loop sequence showing at leastabout 80%, preferably at least about 85%, more preferably at least about90%, or even more preferably at least about 95%, sequence identity withthe not to 100% conserved nucleotides in the histone stem-loop sequencesaccording to at least one of specific formulae (I) or (Ia) to (Ih) or(II) or (IIa) to (IIh) or with a naturally occurring histone stem-loopsequence.

A particular preferred example for a histone stem-loop sequence is thesequence according to SEQ ID NO: 43 (CAAAGGCTCTTTTCAGAGCCACCA) or thecorresponding RNA sequence according to SEQ ID NO.: 44(CAAAGGCUCUUUUCAGAGCCACCA).

Utr Modification:

Preferably, the modified RNA according to the invention has at least onemodified 5′ and/or 3′ UTR sequence (UTR modification). Thesemodifications in the 5′ and/or 3′ untranslated regions (UTR) may havethe effect of increasing the half-life of the RNA in the cytosol or mayincrease the translational efficiency and may thus enhance theexpression of the encoded protein or peptide. These UTR sequences canhave 100% sequence identity to naturally occurring sequences which occurin viruses, bacteria and eukaryotes, but can also be partly orcompletely synthetic. The untranslated sequences (UTR) of the (alpha-)or beta globin gene, e.g. from Homo sapiens or Xenopus laevis may bementioned as an example of stabilizing sequences which can be used for astabilized RNA. Another example of a stabilizing sequence has thegeneral formula (C/U)CCANxCCC(U/A)PyxUC(C/U)CC which is contained in the3′UTR of the very stable RNA which codes for (alpha-)globin,type(I)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf.Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414).Particularly preferred in the context of the present invention is themutated UTR of (alpha-) globin comprising the following sequenceGCCCGaTGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCG (SEQ ID NO. 45) (theunderlined nucleotide shows the mutation compared to the wild typesequence), which is also termed herein as “muag”. Such introduced UTRsequences can, of course, be used individually or in combination withone another and also in combination with other sequence modificationsknown to a person skilled in the art.

Preferably, decay signals are removed from the 3′ untranslated regions(3′ UTRs) of an RNA according to the invention. Specifically, AdenylateUridylate Rich Elements (AREs) are replaced by the 3′UTR of a stablemRNA (e.g. α- or β-globin mRNA) (reviewed in Tavernier et al., J ControlRelease. 2011 Mar. 30; 150(3):238-47. PMID: 20970469).

In this context, it was shown that the 3′UTR of α-globin mRNA may be animportant factor for the well-known stability of α-globin mRNA (Rodgerset al., Regulated α-globin mRNA decay is a cytoplasmic event proceedingthrough 3′-to-5′ exosome-dependent decapping, RNA, 8, pp. 1526-1537,2002). The 3′UTR of α-globin mRNA is apparently involved in theformation of a specific ribonucleoprotein-complex, the α-complex, whosepresence correlates with mRNA stability in vitro (Wang et al., An mRNAstability complex functions with poly(A)-binding protein to stabilizemRNA in vitro, Molecular and Cellular biology, Vol 19, No. 7, July 1999,p. 4552-4560).

In this context, it is particularly preferred that the naturallyoccuring 5′- and/or 3′-UTRs of the gene comprising the open readingframe contained in the modified RNA according to the invention isremoved in the modified RNA. Thus, the modified RNA according to theinvention preferably comprises UTRs which are heterologous to the openreading frame if present in the modified RNA.

In a preferred embodiment, a combination of two or more of themodifications described above are present in one modified RNA that isadministered by jet injection and which comprises at least one openreading frame. In a particularly preferred embodiment, an RNA comprisingat least one open reading frame is modified by introducing a(heterologous) 3′ UTR, a poly(A) tail, a poly(C) sequence and a histonestem-loop. Preferably, the 3′ UTR therein is the mutated UTR of (alpha-)globin comprising the sequence GCCCGaTGGG CCTCCCAACG GGCCCTCCTCCCCTCCTTGC ACCG (SEQ ID NO. 45; muag; the underlined nucleotide showsthe mutation compared to the wild type sequence), the poly(A) tailconsists of 64 adenine nucleotides, the poly(C) sequence consists of 30cytosine nucleotides and the histone stem-loop has a structure selectedfrom one of formulae (I) or (II), preferably the RNA sequence accordingto SEQ ID NO. 44 (CAAAGGCUCUUUUCAGAGCCACCA). Most preferably, the RNA ofthat specific embodiment comprises the sequence modifications as shownin FIG. 4 (SEQ ID NO. 46; see also Example 1).

By a further embodiment, the modified RNA according to the inventionpreferably comprises at least one of the following structural elements:a 5′- and/or 3′-untranslated region element (UTR element), particularlya 5′-UTR element, which comprises or consists of a nucleic acidsequence, which is derived from the 5′-UTR of a TOP gene or from afragment, homolog or a variant thereof, or a 5′- and/or 3′-UTR element,which may be derivable from a gene that provides a stable mRNA or from ahomolog, fragment or variant thereof; a histone-stem-loop structure,preferably a histone-stem-loop in its 3′ untranslated region; a 5′-CAPstructure; a poly-A tail; or a poly(C) sequence.

In a preferred embodiment of the first aspect of the present invention,the modified RNA according to the invention comprises at least one 5′-or 3′-UTR element. In this context an UTR element comprises or consistsof a nucleic acid sequence, which is derived from the 5′- or 3′-UTR ofany naturally occurring gene, or which is derived from a fragment, ahomolog or a variant of the 5′- or 3′-UTR of a gene. Preferably, the 5′-or 3′-UTR element used according to the present invention isheterologous to the coding region of the modified RNA according to theinvention. Even if 5′- or 3′-UTR elements derived from naturallyoccurring genes are preferred, also synthetically engineered UTRelements may be used in the context of the present invention.

In a particularly preferred embodiment of the first aspect of thepresent invention, the sequence of the modified RNA according to theinvention comprises at least one 5′-untranslated region element (5′UTRelement), which comprises or consists of a nucleic acid sequence, whichis derived from the 5′UTR of a TOP gene, or which is derived from afragment, homolog or variant of the 5′UTR of a TOP gene.

It is particularly preferred that the 5′UTR element does not comprise aTOP-motif or a 5′TOP, as defined above.

In some embodiments, the nucleic acid sequence of the 5′UTR element,which is derived from a 5′UTR of a TOP gene terminates at its 3′-endwith a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10upstream of the start codon (e.g. A(U/T)G) of the gene or mRNA it isderived from. Thus, the 5′UTR element does not comprise any part of theprotein coding region. Thus, preferably, the only protein coding part ofthe modified RNA according to the invention is provided by the codingregion.

The nucleic acid sequence, which is derived from the 5′UTR of a TOPgene, is derived from a eukaryotic TOP gene, preferably a plant oranimal TOP gene, more preferably a chordate TOP gene, even morepreferably a vertebrate TOP gene, most preferably a mammalian TOP gene,such as a human TOP gene.

For example, the 5′UTR element is prefereably selected from 5′-UTRelements comprising or consisting of a nucleic acid sequence, which isderived from a nucleic acid sequence selected from the group consistingof SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO.1422 of the patent application WO2013/143700, whose disclosure isincorporated herein by reference, from the homologs of SEQ ID Nos.1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of thepatent application WO2013/143700, from a variant thereof, or preferablyfrom a corresponding RNA sequence. The term “homologs of SEQ ID Nos.1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of thepatent application WO2013/143700” refers to sequences of other speciesthan homo sapiens, which are homologous to the sequences according toSEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422of the patent application WO2013/143700.

In a preferred embodiment, the 5′UTR element comprises or consists of anucleic acid sequence, which is derived from a nucleic acid sequenceextending from nucleotide position 5 (i.e. the nucleotide that islocated at position 5 in the sequence) to the nucleotide positionimmediately 5′ to the start codon (located at the 3′ end of thesequences), e.g. the nucleotide position immediately 5′ to the ATGsequence, of a nucleic acid sequence selected from SEQ ID Nos. 1-1363,SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from the homologs of SEQ ID Nos. 1-1363, SEQID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from a variant thereof, or a correspondingRNA sequence. It is particularly preferred that the 5′ UTR element isderived from a nucleic acid sequence extending from the nucleotideposition immediately 3′ to the 5′TOP to the nucleotide positionimmediately 5′ to the start codon (located at the 3′ end of thesequences), e.g. the nucleotide position immediately 5′ to the ATGsequence, of a nucleic acid sequence selected from SEQ ID Nos. 1-1363,SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from the homologs of SEQ ID Nos. 1-1363, SEQID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from a variant thereof, or a correspondingRNA sequence.

In a particularly preferred embodiment, the 5′UTR element comprises orconsists of a nucleic acid sequence, which is derived from a 5′UTR of aTOP gene encoding a ribosomal protein or from a variant of a 5′UTR of aTOP gene encoding a ribosomal protein. For example, the 5′UTR elementcomprises or consists of a nucleic acid sequence which is derived from a5′UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67,170, 193, 244, 259, 554, 650, 675, 700, 721, 913, 1016, 1063, 1120,1138, and 1284-1360 of the patent application WO2013/143700, acorresponding RNA sequence, a homolog thereof, or a variant thereof asdescribed herein, preferably lacking the 5′TOP motif. As describedabove, the sequence extending from position 5 to the nucleotideimmediately 5′ to the ATG (which is located at the 3′end of thesequences) corresponds to the 5′UTR of said sequences.

Preferably, the 5′UTR element comprises or consists of a nucleic acidsequence, which is derived from a 5′UTR of a TOP gene encoding aribosomal Large protein (RPL) or from a homolog or variant of a 5′UTR ofa TOP gene encoding a ribosomal Large protein (RPL). For example, the5′UTR element comprises or consists of a nucleic acid sequence, which isderived from a 5′UTR of a nucleic acid sequence according to any of SEQID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421 and1422 of the patent application WO2013/143700, a corresponding RNAsequence, a homolog thereof, or a variant thereof as described herein,preferably lacking the 5′TOP motif.

In a particularly preferred embodiment, the 5′UTR element comprises orconsists of a nucleic acid sequence, which is derived from the 5′UTR ofa ribosomal protein Large 32 gene, preferably from a vertebrateribosomal protein Large 32 (L32) gene, more preferably from a mammalianribosomal protein Large 32 (L32) gene, most preferably from a humanribosomal protein Large 32 (L32) gene, or from a variant of the 5′UTR ofa ribosomal protein Large 32 gene, preferably from a vertebrateribosomal protein Large 32 (L32) gene, more preferably from a mammalianribosomal protein Large 32 (L32) gene, most preferably from a humanribosomal protein Large 32 (L32) gene, wherein preferably the 5′UTRelement does not comprise the 5′TOP of said gene.

Accordingly, in a particularly preferred embodiment, the 5′UTR elementcomprises or consists of a nucleic acid sequence which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID No. 55 (5′-UTR ofhuman ribosomal protein Large 32 lacking the 5′ terminal oligopyrimidinetract: GGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATC; corresponding to SEQID No. 1368 of the patent application WO2013/143700) or preferably to acorresponding RNA sequence, or wherein the at least one 5′UTR elementcomprises or consists of a fragment of a nucleic acid sequence which hasan identity of at least about 40%, preferably of at least about 50%,preferably of at least about 60%, preferably of at least about 70%, morepreferably of at least about 80%, more preferably of at least about 90%,even more preferably of at least about 95%, even more preferably of atleast about 99% to the nucleic acid sequence according to SEQ ID No. 55or more preferably to a corresponding RNA sequence, wherein, preferably,the fragment is as described above, i.e. being a continuous stretch ofnucleotides representing at least 20% etc. of the full-length 5′UTR.Preferably, the fragment exhibits a length of at least about 20nucleotides or more, preferably of at least about 30 nucleotides ormore, more preferably of at least about 40 nucleotides or more.Preferably, the fragment is a functional fragment as described herein.

In some embodiments, the modified RNA according to the inventioncomprises a 5′UTR element, which comprises or consists of a nucleic acidsequence, which is derived from the 5′UTR of a vertebrate TOP gene, suchas a mammalian, e.g. a human TOP gene, selected from RPSA, RPS2, RPS3,RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13,RPS14, RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23,RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4,RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13,RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23,RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29, RPL30, RPL31, RPL32,RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37A, RPL38, RPL39, RPL40,RPL41, RPLP0, RPLP1, RPLP2, RPLP3, RPLP0, RPLP1, RPLP2, EEF1A1, EEF1B2,EEF1D, EEF1G, EEF2, EIF3E, EIF3F, EIF3H, EIF2S3, EIF3C, EIF3K, EIF3EIP,EIF4A2, PABPC1, HNRNPA1, TPT1, TUBB1, UBA52, NPM1, ATP5G2, GNB2L1, NME2,UQCRB, or from a homolog or variant thereof, wherein preferably the5′UTR element does not comprise a TOP-motif or the 5′TOP of said genes,and wherein optionally the 5′UTR element starts at its 5′-end with anucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10downstream of the 5′terminal oligopyrimidine tract (TOP), and whereinfurther optionally the 5′UTR element, which is derived from a 5′UTR of aTOP gene, terminates at its 3′-end with a nucleotide located at position1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of the start codon (A(U/T)G) ofthe gene it is derived from.

In further particularly preferred embodiments, the 5′UTR elementcomprises or consists of a nucleic acid sequence, which is derived fromthe 5′UTR of a ribosomal protein Large 32 gene (RPL32), a ribosomalprotein Large 35 gene (RPL35), a ribosomal protein Large 21 gene(RPL21), an ATP synthase, H+ transporting, mitochondrial F1 complex,alpha subunit 1, cardiac muscle (ATP5A1) gene, an hydroxysteroid(17-beta) dehydrogenase 4 gene (HSD17B4), an androgen-induced 1 gene(AIG1), cytochrome c oxidase subunit VIc gene (COX6C), or aN-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, preferably from a vertebrate ribosomal proteinLarge 32 gene (RPL32), a vertebrate ribosomal protein Large 35 gene(RPL35), a vertebrate ribosomal protein Large 21 gene (RPL21), avertebrate ATP synthase, H+ transporting, mitochondrial F1 complex,alpha subunit 1, cardiac muscle (ATP5A1) gene, a vertebratehydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4), a vertebrateandrogen-induced 1 gene (AIG1), a vertebrate cytochrome c oxidasesubunit VIc gene (COX6C), or a vertebrate N-acylsphingosineamidohydrolase (acid ceramidase) 1 gene (ASAH1) or from a variantthereof, more preferably from a mammalian ribosomal protein Large 32gene (RPL32), a ribosomal protein Large 35 gene (RPL35), a ribosomalprotein Large 21 gene (RPL21), a mammalian ATP synthase, H+transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle(ATP5A1) gene, a mammalian hydroxysteroid (17-beta) dehydrogenase 4 gene(HSD17B4), a mammalian androgen-induced 1 gene (AIG1), a mammaliancyto-chrome c oxidase subunit VIc gene (COX6C), or a mammalianN-acylsphingosine ami-dohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, most preferably from a human ribosomal proteinLarge 32 gene (RPL32), a human ribosomal protein Large 35 gene (RPL35),a human ribosomal protein Large 21 gene (RPL21), a human ATP syn-thase,H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiacmuscle (ATP5A1) gene, a human hydroxysteroid (17-beta) dehydrogenase 4gene (HSD17B4), a human androgen-induced 1 gene (AIG1), a humancytochrome c oxidase subunit VIc gene (COX6C), or a humanN-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, wherein preferably the 5′UTR element does notcomprise the 5′TOP of said gene.

In a particularly preferred embodiment, the 5′UTR element comprises orconsists of a nucleic acid sequence, which is derived from the 5′UTR ofan hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4), preferablyfrom a vertebrate hydroxysteroid (17-beta) dehydrogenase 4 gene(HSD17B4), more preferably from a mammalian hydroxysteroid (17-beta)dehydrogenase 4 gene (HSD17B4), most preferably from a humanhydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4). In a preferredembodiment, the 5′UTR element comprises or consists of a nucleic acidsequence that has an identity of at least about 40%, preferably of atleast about 50%, preferably of at least about 60%, preferably of atleast about 70%, more preferably of at least about 80%, more preferablyof at least about 90%, even more preferably of at least about 95%, evenmore preferably of at least about 99% to the 5′UTR comprised in thenucleic acid sequence according to SEQ ID NO: 54. Preferably, the 5′UTRcomprises or consists of the 5′UTR as comprised in the nucleic acidsequence according to SEQ ID NO: 54.

Accordingly, in a particularly preferred embodiment, the 5′UTR elementcomprises or consists of a nucleic acid sequence, which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID No. 1368, or SEQ IDNOs 1412-1420 of the patent application WO2013/143700, or acorresponding RNA sequence, or wherein the at least one 5′UTR elementcomprises or consists of a fragment of a nucleic acid sequence, whichhas an identity of at least about 40%, preferably of at least about 50%,preferably of at least about 60%, preferably of at least about 70%, morepreferably of at least about 80%, more preferably of at least about 90%,even more preferably of at least about 95%, even more preferably of atleast about 99% to the nucleic acid sequence according to SEQ ID No.1368, or SEQ ID NOs 1412-1420 of the patent application WO2013/143700,wherein, preferably, the fragment is as described above, i.e. being acontinuous stretch of nucleotides representing at least 20% etc. of thefull-length 5′UTR. Preferably, the fragment exhibits a length of atleast about 20 nucleotides or more, preferably of at least about 30nucleotides or more, more preferably of at least about 40 nucleotides ormore. Preferably, the fragment is a functional fragment as describedherein.

Accordingly, in a particularly preferred embodiment, the 5′UTR elementcomprises or consists of a nucleic acid sequence, which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID No. 56 (5′-UTR ofATP5A1 lacking the 5′ terminal oligopyrimidine tract:GCGGCTCGGCCATTTTGTCCCAGTCAGTCCGGAGGCTGCGGCTGCAGAAGTACCGCCTGCG-GAGTAACTGCAAAG; corresponding to SEQ ID No. 1414 of the patentapplication WO2013/143700) or preferably to a corresponding RNAsequence, or wherein the at least one 5′UTR element comprises orconsists of a fragment of a nucleic acid sequence, which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID No. 26 or morepreferably to a corresponding RNA sequence, wherein, preferably, thefragment is as described above, i.e. being a continuous stretch ofnucleotides representing at least 20% etc. of the full-length 5′UTR.Preferably, the fragment exhibits a length of at least about 20nucleotides or more, preferably of at least about 30 nucleotides ormore, more preferably of at least about 40 nucleotides or more.Preferably, the fragment is a functional fragment as described herein.

In a further preferred embodiment, the modified RNA according to theinvention further comprises at least one 3′UTR element, which comprisesor consists of a nucleic acid sequence derived from the 3′UTR of achordate gene, preferably a vertebrate gene, more preferably a mammaliangene, most preferably a human gene, or from a variant of the 3′UTR of achordate gene, preferably a vertebrate gene, more preferably a mammaliangene, most preferably a human gene.

The term ‘3′UTR element’ refers to a nucleic acid sequence, whichcomprises or consists of a nucleic acid sequence that is derived from a3′UTR or from a variant of a 3′UTR. A 3′UTR element in the sense of thepresent invention may represent the 3′UTR of an mRNA. Thus, in the senseof the present invention, preferably, a 3′UTR element may be the 3′UTRof an mRNA, preferably of an artificial mRNA, or it may be thetranscription template for a 3′UTR of an mRNA. Thus, a 3′UTR elementpreferably is a nucleic acid sequence, which corresponds to the 3′UTR ofan mRNA, preferably to the 3′UTR of an artificial mRNA, such as an mRNAobtained by transcription of a genetically engineered vector construct.Preferably, the 3′UTR element fulfils the function of a 3′UTR or encodesa sequence, which fulfils the function of a 3′UTR.

Preferably, the inventive mRNA comprises a 3′UTR element, which may bederivable from a gene that relates to an mRNA with an enhanced half-life(that provides a stable mRNA), for example a 3′UTR element as definedand described below.

In a particularly preferred embodiment, the 3′UTR element comprises orconsists of a nucleic acid sequence, which is derived from a 3′UTR of agene selected from the group consisting of an albumin gene, an α-globingene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene,and a collagen alpha gene, such as a collagen alpha 1(I) gene, or from avariant of a 3′UTR of a gene selected from the group consisting of analbumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylasegene, a lipoxygenase gene, and a collagen alpha gene, such as a collagenalpha 1(I) gene according to SEQ ID No. 1369-1390 of the patentapplication WO2013/143700, whose disclosure is incorporated herein byreference. In a particularly preferred embodiment, the 3′UTR elementcomprises or consists of a nucleic acid sequence, which is derived froma 3′UTR of an albumin gene, preferably a vertebrate albumin gene, morepreferably a mammalian albumin gene, most preferably a human albumingene according to SEQ ID No. 57.

Human albumin 3′UTR SEQ ID No. 57:

CATCACATTT AAAAGCATCT CAGCCTACCA TGAGAATAAG AGAAAGAAAA TGAAGATCAAAAGCTTATTC ATCTGTTTTT CTTTTTCGTT GGTGTAAAGC CAACACCCTG TCTAAAAAACATAAATTTCT TTAATCATTT TGCCTCTTTT CTCTGTGCTT CAATTAATAA AAAATGGAAA GAATCT(corresponding to SEQ ID No: 1369 of the patent applicationWO2013/143700).

In this context, it is particularly preferred that the modified RNAaccording to the invention comprises a 3′-UTR element comprising acorresponding RNA sequence derived from the nucleic acids according toSEQ ID No. 1369-1390 of the patent application WO2013/143700 or afragment, homolog or variant thereof.

Most preferably the 3′-UTR element comprises the nucleic acid sequencederived from a fragment of the human albumin gene according to SEQ IDNo. 58:

albumin7 3′UTR CATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCT. (SEQ ID No. 58 correspondingto SEQ ID No: 1376 of the patent application WO2013/143700)

In this context, it is particularly preferred that the 3′-UTR element ofthe inventive mRNA comprises or consists of a corresponding RNA sequenceof the nucleic acid sequence according to SEQ ID No. 58.

In another particularly preferred embodiment, the 3′UTR elementcomprises or consists of a nucleic acid sequence, which is derived froma 3′UTR of an α-globin gene, preferably a vertebrate α- or β-globingene, more preferably a mammalian α- or β-globin gene, most preferably ahuman α- or β-globin gene according to SEQ ID No. 59-61:

3′-UTR of Homo sapiens hemoglobin, alpha 1 (HBA1)GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAA GTCTGAGTGGGCGGC (SEQ IDNo: 59 corresponding to SEQ ID No. 1370 of the patent applicationWO2013/143700) 3′-UTR of Homo sapiens hemoglobin, alpha 2 (HBA2)GCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTGAATAAAG TCTGAGTGGGCAG (SEQ IDNo: 60 corresponding to SEQ ID No. 1371 of the patent applicationWO013/143700) 3′-UTR of Homo sapiens hemoglobin, beta (HBB)GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC (SEQ ID No: 61 corresponding to SEQID No. 1372 of the patent application WO2013/143700)

For example, the 3′UTR element may comprise or consist of the center,α-complex-binding portion of the 3′UTR of an α-globin gene, such as of ahuman α-globin gene, preferably according to SEQ ID No. 62:

Center, α-complex-binding portion of the 3′UTR of an α-globin gene (alsonamed herein as “muag”) GCCCGATGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCG(SEQ ID NO. 62 corresponding to SEQ ID No. 1393 of the patentapplication WO2013/143700).

In this context, it is particularly preferred that the 3′-UTR element ofthe modified RNA according to the invention comprises or consists of acorresponding RNA sequence of the nucleic acid sequence according to SEQID No. 62 or a homolog, a fragment or variant thereof.

The term ‘a nucleic acid sequence, which is derived from the 3′UTR of a[ . . . ] gene’ preferably refers to a nucleic acid sequence, which isbased on the 3′UTR sequence of a [ . . . ] gene or on a part thereof,such as on the 3′UTR of an albumin gene, an α-globin gene, a 3-globingene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagenalpha gene, such as a collagen alpha 1(I) gene, preferably of an albumingene or on a part thereof. This term includes sequences corresponding tothe entire 3′UTR sequence, i.e. the full length 3′UTR sequence of agene, and sequences corresponding to a fragment of the 3′UTR sequence ofa gene, such as an albumin gene, α-globin gene, -globin gene, tyrosinehydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as acollagen alpha 1(I) gene, preferably of an albumin gene.

The term ‘a nucleic acid sequence, which is derived from a variant ofthe 3′UTR of a [ . . . ] gene’ preferably refers to a nucleic acidsequence, which is based on a variant of the 3′UTR sequence of a gene,such as on a variant of the 3′UTR of an albumin gene, an α-globin gene,a 3-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, or acollagen alpha gene, such as a collagen alpha 1(I) gene, or on a partthereof as described above. This term includes sequences correspondingto the entire sequence of the variant of the 3′UTR of a gene, i.e. thefull length variant 3′UTR sequence of a gene, and sequencescorresponding to a fragment of the variant 3′UTR sequence of a gene. Afragment in this context preferably consists of a continuous stretch ofnucleotides corresponding to a continuous stretch of nucleotides in thefull-length variant 3′UTR, which represents at least 20%, preferably atleast 30%, more preferably at least 40%, more preferably at least 50%,even more preferably at least 60%, even more preferably at least 70%,even more preferably at least 80%, and most preferably at least 90% ofthe full-length variant 3′UTR. Such a fragment of a variant, in thesense of the present invention, is preferably a functional fragment of avariant as described herein.

Preferably, the at least one 5′UTR element and the at least one 3′UTRelement act synergistically to increase protein production from themodified RNA according to the invention as described above. Morepreferably, the at least one 5′UTR element and/or the at least one 3′UTRelement act synergistically together with jet injection of the modifiedRNA in order to increase protein production.

In further specific embodiments, the modified RNA according to theinvention may—in addition to the at least one modification—furthercomprise an internal ribosome entry site (IRES) sequence or IRES-motif,which may separate several open reading frames, for example if themodified RNA encodes for two or more peptides or proteins. AnIRES-sequence may be particularly helpful if the mRNA is a bi- ormulticistronic RNA.

As used in the context of the present invention, the term “RNA” is notlimiting and refers to any ribonucleic acid. Thus, the term RNA equallyapplies to ribonucleic acids that function as coding RNAs for example,as viral RNA, replicon or as mRNA or to artificial RNA constructs of anytype.

In a preferred embodiment, the modified RNA is not a small interferingRNA (siRNA) or a short hairpin RNA (shRNA). Preferably, the modified RNAis not an siRNA or shRNA against a resistance gene, preferably amultidrug resistance gene, such as MDR1/P-gp.

According to the invention, the RNA comprises at least one modification,which increases the expression of the peptide or protein encoded by thecoding region of the RNA. In a preferred embodiment, the RNA accordingto the invention comprises at least one type of modification as definedherein. In another preferred embodiment, the RNA according to theinvention comprises at least two distinct types of modifications thatincrease the expression of the peptide or protein encoded by the codingregion of the RNA. Preferably, the RNA according to the inventioncomprises at least two, three, four, five, six, seven, eight, nine, ten,eleven or twelve distinct types of modifications. Alternatively, or incombination with other other types of modifications, a specificmodification as defined herein, such as a 3′UTR, a histone stem-loop ora poly C sequence, may also be present in more than one copy in themodified RNA according to the invention. This holds true in particularfor bi- or multicistronic RNAs. A modified RNA according to theinvention may comprise combinations of several distinct modifications,such as GC-enrichment in combination with any of, for instance, a 5′UTR,a poly C sequence, a poly A sequence, a 3′UTR or a histone stem-loop,wherein each distinct modification may be present in the form of asingle copy per RNA or in the form of multiple copies per RNA.

Preferred embodiments of the RNA according to the invention maytherefore comprise, for example,

5′-coding region-histone stem-loop-3′;5′-coding region-histone stem-loop-poly(A)/(C) sequence-3′; or5′-coding region-poly(A)/(C) sequence-histone stem-loop-3′; or5′-coding region-histone stem-loop-polyadenylation signal-3′; or5′-coding region-polyadenylation signal-histone stem-loop-3′; or5′-coding region-histone stem-loop-histone stem-loop-poly(A)/(C)sequence-3′; or5′-coding region-histone stem-loop-histone stem-loop-polyadenylationsignal-3′; or5′-coding region-poly(A)/(C) sequence-poly(A)/(C) sequence-histonestem-loop-3′; etc.,wherein the coding region preferably comprises or consists of a GCenriched sequence as defined herein, andwherein, a 5′UTR element is preferably present on the 5′ side of thecoding region and/or a 3′UTR element is preferably present between the3′ end of the coding region and the 3′ terminus of the RNA.

A specific embodiment of the modified RNA may comprise, for instance,any one of the combinations of structural features and modifications aslisted below:

5′-coding region-poly(A) sequence-3′;5′-coding region-poly(A) sequence-poly(C) sequence-3′;5′-coding region-poly(A) sequence-poly(C) sequence-histone stem-loop-3′;5′-coding region-3′UTR-poly(A) sequence-3′;5′-coding region-3′UTR-poly(A) sequence-poly(C) sequence-3′;5′-coding region-3′UTR-poly(A) sequence-poly(C) sequence-histonestem-loop-3′;5′-5′UTR-coding region-poly(A) sequence-3′;5′-5′UTR-coding region-poly(A) sequence-poly(C) sequence-3′;5′-5′UTR-coding region-poly(A) sequence-poly(C) sequence-histonestem-loop-3′;5′-coding region-3′UTR-poly(A) sequence-3′;5′-5′UTR-coding region-3′UTR-poly(A) sequence-poly(C) sequence-3′;5′-5′UTR-coding region-3′UTR-poly(A) sequence-poly(C) sequence-histonestem-loop-3′;wherein the coding region preferably comprises or consists of a GCenriched sequence as defined herein.

Encoded Proteins:

In a preferred embodiment, the modified RNA comprises at least one openreading frame, which encodes a therapeutic protein or peptide. Inanother embodiment, an antigen is encoded by the at least one openreading frame, such as a pathogenic antigen, a tumour antigen, anallergenic antigen or an autoimmune antigen. Therein, the administrationof the modified RNA encoding the antigen is used in a geneticvaccination approach against a disease involving said antigen.

In an alternative embodiment, an antibody is encoded by the at least oneopen reading frame of the modified RNA according to the invention.

In a preferred embodiment, the modified RNA according to the inventiondoes not comprise a reporter gene or a marker gene. Preferably, themodified RNA according to the invention does not encode, for instance,luciferase; green fluorescent protein (GFP) and its variants (such aseGFP, RFP or BFP); α-globin; hypoxanthine-guaninephosphoribosyltransferase (HGPRT); β-galactosidase; galactokinase;alkaline phosphatase; secreted embryonic alkaline phosphatase (SEAP)) ora resistance gene (such as a resistance gene against neomycin,puromycin, hygromycin and zeocin). In a preferred embodiment, themodified RNA according to the invention does not encode luciferase. Inanother embodiment, the modified RNA according to the invention does notencode GFP or a variant thereof.

Antigens: Pathogenic Antigens:

The modified RNA according to the present invention may encode a proteinor a peptide, which comprises a pathogenic antigen or a fragment,variant or derivative thereof. Such pathogenic antigens are derived frompathogenic organisms, in particular bacterial, viral or protozoological(multicellular) pathogenic organisms, which evoke an immunologicalreaction in a subject, in particular a mammalian subject, moreparticularly a human. More specifically, pathogenic antigens arepreferably surface antigens, e.g. proteins (or fragments of proteins,e.g. the exterior portion of a surface antigen) located at the surfaceof the virus or the bacterial or protozoological organism.

Pathogenic antigens are peptide or protein antigens preferably derivedfrom a pathogen associated with infectious disease, which are preferablyselected from antigens derived from the pathogens Acinetobacterbaumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostomabraziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum,Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus,Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus,Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis,Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus,Brugia malayi, Bunyaviridae family, Burkholderia cepacia and otherBurkholderia species, Burkholderia mallei, Burkholderia pseudomallei,Caliciviridae family, Campylobacter genus, Candida albicans, Candidaspp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophilapsittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum,Clostridium difficile, Clostridium perfringens, Clostridium perfringens,Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses,Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congohemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus,Cytomegalovirus (CMV), Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4),Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichiachaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica,Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie Avirus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus(EBV), Escherichia coli O157:H7, 0111 and 0104:H4, Fasciola hepatica andFasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses,Francisella tularensis, Fusobacterium genus, Geotrichum candidum,Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus,Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori,Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis BVirus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis EVirus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasmacapsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Humanbocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7(HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Humanparainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus,Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassavirus, Legionella pneumophila, Leishmania genus, Leptospira genus,Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV),Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimusyokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV),Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis,Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasmapneumoniae, Naegleria fowleri, Necator americanus, Neisseriagonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp,Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family(Influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimuswestermani, Parvovirus B19, Pasteurella genus, Plasmodium genus,Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory syncytialvirus (RSV), Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsiagenus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi,Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus,Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosomagenus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrixschenckii, Staphylococcus genus, Staphylococcus genus, Streptococcusagalactiae, Streptococcus pneumoniae, Streptococcus pyogenes,Strongyloides stercoralis, Taenia genus, Taenia solium, Tick-borneencephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasmagondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis,Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosomacruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Varicellazoster virus (VZV), Variola major or Variola minor, vCJD prion,Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus,Western equine encephalitis virus, Wuchereria bancrofti, Yellow fevervirus, Yersinia enterocolitica, Yersinia pestis, and Yersiniapseudotuberculosis.

In this context particularly preferred are antigens from the pathogensselected from Influenza virus, respiratory syncytial virus (RSV), Herpessimplex virus (HSV), human Papilloma virus (HPV), Human immunodeficiencyvirus (HIV), Plasmodium, Staphylococcus aureus, Dengue virus, Chlamydiatrachomatis, Cytomegalovirus (CMV), Hepatitis B virus (HBV),Mycobacterium tuberculosis, Rabies virus, and Yellow Fever Virus.

In a preferred embodiment, the modified RNA according to the inventionencodes a Rabies virus protein or peptide or an antigenic fragmentthereof. Preferably, the modified RNA according to the invention encodesan antigenic protein or peptide selected from the group consisting ofglycoprotein G (RAV-G), nucleoprotein N (RAV-N), phosphoprotein P(RAV-P), matrix protein M (RAV-M) or RNA polymerase L (RAV-L) of Rabiesvirus, or a fragment, variant or derivative thereof.

In another preferred embodiment, the modified RNA according to theinvention encodes a respiratory syncytial virus (RSV) protein or peptideor an antigenic fragment thereof. Preferably, the modified RNA accordingto the invention encodes an antigenic protein or peptide selected fromthe group consisting of the fusion protein F, the glycoprotein G, theshort hydrophobic protein SH, the matrix protein M, the nucleoprotein N,the large polymerase L, the M2-1 protein, the M2-2 protein, thephosphoprotein P, the non-structural protein NS1 or the non-structuralprotein NS2 of respiratory syncytial virus (RSV), or a fragment, variantor derivative thereof.

Tumour Antigens:

In a further embodiment, the modified RNA according to the presentinvention may encode a protein or a peptide, which comprises a peptideor protein comprising a tumour antigen, a fragment, variant orderivative of said tumour antigen, preferably, wherein the tumourantigen is a melanocyte-specific antigen, a cancer-testis antigen or atumour-specific antigen, preferably a CT-X antigen, a non-X CT-antigen,a binding partner for a CT-X antigen or a binding partner for a non-XCT-antigen or a tumour-specific antigen, more preferably a CT-X antigen,a binding partner for a non-X CT-antigen or a tumour-specific antigen ora fragment, variant or derivative of said tumour antigen; and whereineach of the nucleic acid sequences encodes a different peptide orprotein; and wherein at least one of the nucleic acid sequences encodesfor 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin,alpha-5-beta-6-integrin, alpha-actinin-4/m, alpha-methylacyl-coenzyme Aracemase, ART-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m,BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125,calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20,CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m,CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m, coactosin-like protein,collage XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B,CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam,EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1,GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V,gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL,HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE, homeobox NKX3.1,HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT,iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor,kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m, KK-LC-1,K-Ras/m, LAGE-A, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6,MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4,MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2,MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1,MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrix protein22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen,MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m,NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m,NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP,OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190 minorbcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF,Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein,proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m,RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC,SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1,survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP,TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1,TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, WT1and a immunoglobulin idiotype of a lymphoid blood cell or a T cellreceptor idiotype of a lymphoid blood cell, or a fragment, variant orderivative of said tumour antigen; preferably survivin or a homologuethereof, an antigen from the MAGE-family or a binding partner thereof ora fragment, variant or derivative of said tumour antigen.

Particularly preferred in this context are the tumour antigens NY-ESO-1,5T4, MAGE-C1, MAGE-C2, Survivin, Muc-1, PSA, PSMA, PSCA, STEAP and PAP.

In this context, it is particularly preferred that at least one modifiedRNA administered by jet injection according to the invention encodes oneof the following combinations of antigens:

-   -   Muc-1, PSA, PSMA, PSCA, and STEAP    -   Muc-1, PSA, PSMA, PSCA, and PAP    -   Muc-1, PSA, PSMA, STEAP and PAP    -   Muc-1, PSA, PSCA, STEAP and PAP    -   Muc-1, PSMA, PSCA, STEAP and PAP    -   PSA, PSMA, PSCA, STEAP and PAP    -   Muc-1, PSA, PSMA, PSCA, STEAP and PAP

In another embodiment, it is particularly preferred that at least onemodified RNA administered by jet injection according to the inventionencodes one of the following combinations of antigens:

-   -   NY-ESO-1, 5T4, MAGE-C1, MAGE-C2, and Survivin    -   NY-ESO-1, 5T4, MAGE-C1, MAGE-C2, and Muc-1    -   NY-ESO-1, 5T4, MAGE-C1, Survivin and Muc-1    -   NY-ESO-1, 5T4, MAGE-C2, Survivin and Muc-1    -   NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and Muc-1    -   5T4, MAGE-C1, MAGE-C2, Survivin and Muc-1    -   NY-ESO-1, 5T4, MAGE-C1, MAGE-C2, Survivin and Muc-1

In a preferred embodiment, the modified RNA administered by jetinjection encodes a protein or a peptide, which comprises a therapeuticprotein or a fragment, variant or derivative thereof.

Therapeutic proteins as defined herein are peptides or proteins, whichare beneficial for the treatment of any inherited or acquired disease,or which improves the condition of an individual. Particularly,therapeutic proteins play a big role in the creation of therapeuticagents that could modify and repair genetic errors, destroy cancer cellsor pathogen infected cells, treat immune system disorders, treatmetabolic or endocrine disorders, among other functions. For instance,Erythropoietin (EPO), a protein hormone can be utilized in treatingpatients with erythrocyte deficiency, which is a common cause of kidneycomplications. Furthermore adjuvant proteins, therapeutic antibodies areencompassed by therapeutic proteins and also hormone replacementtherapy, which is e.g. used in the therapy of women in menopause. Inmore recent approaches, somatic cells of a patient are used to reprogramthem into pluripotent stem cells, which replace the disputed stem celltherapy. Also these proteins used for reprogramming of somatic cells orused for differentiating of stem cells are defined herein as therapeuticproteins. Furthermore, therapeutic proteins may be used for otherpurposes, e.g. wound healing, tissue regeneration, angiogenesis, etc.

Therefore therapeutic proteins can be used for various purposesincluding treatment of various diseases like e.g. infectious diseases,neoplasms (e.g. cancer or tumour diseases), diseases of the blood andblood-forming organs, endocrine, nutritional and metabolic diseases,diseases of the nervous system, diseases of the circulatory system,diseases of the respiratory system, diseases of the digestive system,diseases of the skin and subcutaneous tissue, diseases of themusculoskeletal system and connective tissue, and diseases of thegenitourinary system, independently if they are inherited or acquired.

In this context, particularly preferred therapeutic proteins which canbe used inter alia in the treatment of metabolic or endocrine disordersare selected from: Acid sphingomyelinase (Niemann-Pick disease),Adipotide (obesity), Agalsidase-beta (human galactosidase A) (Fabrydisease; prevents accumulation of lipids that could lead to renal andcardiovascular complications), Alglucosidase (Pompe disease (glycogenstorage disease type II)), alpha-galactosidase A (alpha-GAL A,Agalsidase alpha) (Fabry disease), alpha-glucosidase (Glycogen storagedisease (GSD), Morbus Pompe), alpha-L-iduronidase (mucopolysaccharidoses(MPS), Hurler syndrome, Scheie syndrome), alpha-N-acetylglucosaminidase(Sanfilippo syndrome), Amphiregulin (cancer, metabolic disorder),Angiopoietin ((Ang1, Ang2, Ang3, Ang4, ANGPTL2, ANGPTL3, ANGPTL4,ANGPTL5, ANGPTL6, ANGPTL7) (angiogenesis, stabilize vessels),Betacellulin (metabolic disorder), Beta-glucuronidase (Sly syndrome),Bone morphogenetic protein BMPs (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6,BMP7, BMP8a, BMP8b, BMP10, BMP15) (regenerative effect, bone-relatedconditions, chronic kidney disease (CKD)), CLN6 protein (CLN6disease—Atypical Late Infantile, Late Onset variant, Early Juvenile,Neuronal Ceroid Lipofuscinoses (NCL)), Epidermal growth factor (EGF)(wound healing, regulation of cell growth, proliferation, anddifferentiation), Epigen (metabolic disorder), Epiregulin (metabolicdisorder), Fibroblast Growth Factor (FGF, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, FGF-16, FGF-17, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22,FGF-23) (wound healing, angiogenesis, endocrine disorders, tissueregeneration), Galsulphase (Mucopolysaccharidosis VI), Ghrelin(irritable bowel syndrome (IBS), obesity, Prader-Willi syndrome, type IIdiabetes mellitus), Glucocerebrosidase (Gaucher's disease), GM-CSF(regenerative effect, production of white blood cells, cancer),Heparin-binding EGF-like growth factor (HB-EGF) (wound healing, cardiachypertrophy and heart development and function), Hepatocyte growthfactor HGF (regenerative effect, wound healing), Hepcidin (ironmetabolism disorders, Beta-thalassemia), Human albumin (Decreasedproduction of albumin (hypoproteinaemia), increased loss of albumin(nephrotic syndrome), hypovolaemia, hyperbilirubinaemia), Idursulphase(Iduronate-2-sulphatase) (Mucopolysaccharidosis II (Hunter syndrome)),Integrins αVβ, αVβ5 and α5β1 (Bind matrix macromolecules andproteinases, angiogenesis), luduronate sulfatase (Hunter syndrome),Laronidase (Hurler and Hurler-Scheie forms of mucopolysaccharidosis I),N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase, Arylsulfatase A(ARSA), Arylsulfatase B (ARSB)) (arylsulfatase B deficiency,Maroteaux-Lamy syndrome, mucopolysaccharidosis VI),N-acetylglucosamine-6-sulfatase (Sanfilippo syndrome), Nerve growthfactor (NGF, Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3(NT-3), and Neurotrophin 4/5 (NT-4/5) (regenerative effect,cardiovascular diseases, coronary atherosclerosis, obesity, type 2diabetes, metabolic syndrome, acute coronary syndromes, dementia,depression, schizophrenia, autism, Rett syndrome, anorexia nervosa,bulimia nervosa, wound healing, skin ulcers, corneal ulcers, Alzheimer'sdisease), Neuregulin (NRG1, NRG2, NRG3, NRG4) (metabolic disorder,schizophrenia), Neuropilin (NRP-1, NRP-2) (angiogenesis, axon guidance,cell survival, migration), Obestatin (irritable bowel syndrome (IBS),obesity, Prader-Willi syndrome, type II diabetes mellitus), PlateletDerived Growth factor (PDGF (PDFF-A, PDGF-B, PDGF-C, PDGF-D)(regenerative effect, wound healing, disorder in angiogenesis,Arteriosclerosis, Fibrosis, cancer), TGF beta receptors (endoglin,TGF-beta 1 receptor, TGF-beta 2 receptor, TGF-beta 3 receptor) (renalfibrosis, kidney disease, diabetes, ultimately end-stage renal disease(ESRD), angiogenesis), Thrombopoietin (THPO) (Megakaryocyte growth anddevelopment factor (MGDF)) (platelets disorders, platelets for donation,recovery of platelet counts after myelosuppressive chemotherapy),Transforming Growth factor (TGF (TGF-a, TGF-beta (TGFbeta1, TGFbeta2,and TGFbeta3))) (regenerative effect, wound healing, immunity, cancer,heart disease, diabetes, Marfan syndrome, Loeys-Dietz syndrome), VEGF(VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F und PIGF) (regenerativeeffect, angiogenesis, wound healing, cancer, permeability), Nesiritide(Acute decompensated congestive heart failure), Trypsin (Decubitusulcer, varicose ulcer, debridement of eschar, dehiscent wound, sunburn,meconium ileus), adrenocorticotrophic hormone (ACTH) (“Addison'sdisease, Small cell carcinoma, Adrenoleukodystrophy, Congenital adrenalhyperplasia, Cushing's syndrome, Nelson's syndrome, Infantile spasms),Atrial-natriuretic peptide (ANP) (endocrine disorders), Cholecystokinin(diverse), Gastrin (hypogastrinemia), Leptin (Diabetes,hypertriglyceridemia, obesity), Oxytocin (stimulate breastfeeding,non-progression of parturition), Somatostatin (symptomatic treatment ofcarcinoid syndrome, acute variceal bleeding, and acromegaly, polycysticdiseases of the liver and kidney, acromegaly and symptoms caused byneuroendocrine tumors), Vasopressin (antidiuretic hormone) (diabetesinsipidus), Calcitonin (Postmenopausal osteoporosis, Hypercalcaemia,Paget's disease, Bone metastases, Phantom limb pain, Spinal Stenosis),Exenatide (Type 2 diabetes resistant to treatment with metformin and asulphonylurea), Growth hormone (GH), somatotropin (Growth failure due toGH deficiency or chronic renal insufficiency, Prader-Willi syndrome,Turner syndrome, AIDS wasting or cachexia with antiviral therapy),Insulin (Diabetes mellitus, diabetic ketoacidosis, hyperkalaemia),Insulin-like growth factor 1 IGF-1 (Growth failure in children with GHgene deletion or severe primary IGF1 deficiency, neurodegenerativedisease, cardiovascular diseases, heart failure), Mecasermin rinfabate,IGF-1 analog (Growth failure in children with GH gene deletion or severeprimary IGF1 deficiency, neurodegenerative disease, cardiovasculardiseases, heart failure), Mecasermin, IGF-1 analog (Growth failure inchildren with GH gene deletion or severe primary IGF1 deficiency,neurodegenerative disease, cardiovascular diseases, heart failure),Pegvisomant (Acromegaly), Pramlintide (Diabetes mellitus, in combinationwith insulin), Teriparatide (human parathyroid hormone residues 1-34)(Severe osteoporosis), Becaplermin (Debridement adjunct for diabeticulcers), Dibotermin-alpha (Bone morphogenetic protein 2) (Spinal fusionsurgery, bone injury repair), Histrelin acetate (gonadotropin releasinghormone; GnRH) (Precocious puberty), Octreotide (Acromegaly, symptomaticrelief of VIP-secreting adenoma and metastatic carcinoid tumours), andPalifermin (keratinocyte growth factor; KGF) (Severe oral mucositis inpatients undergoing chemotherapy, wound healing). (in brackets is theparticular disease for which the therapeutic protein is used in thetreatment). These and other proteins are understood to be therapeutic,as they are meant to treat the subject by replacing its defectiveendogenous production of a functional protein in sufficient amounts.Accordingly, such therapeutic proteins are typically mammalian, inparticular human proteins.

For the treatment of blood disorders, diseases of the circulatorysystem, diseases of the respiratory system, cancer or tumour diseases,infectious diseases or immunedeficiencies following therapeutic proteinsmay be used: Alteplase (tissue plasminogen activator; tPA) (Pulmonaryembolism, myocardial infarction, acute ischaemic stroke, occlusion ofcentral venous access devices), Anistreplase (Thrombolysis),Antithrombin III (AT-III) (Hereditary AT-III deficiency,Thromboembolism), Bivalirudin (Reduce blood-clotting risk in coronaryangioplasty and heparin-induced thrombocytopaenia), Darbepoetin-alpha(Treatment of anaemia in patients with chronic renal insufficiency andchronic renal failure (+/−dialysis)), Drotrecogin-alpha (activatedprotein C) (Severe sepsis with a high risk of death), Erythropoietin,Epoetin-alpha, erythropoetin, erthropoyetin (Anaemia of chronic disease,myleodysplasia, anaemia due to renal failure or chemotherapy,preoperative preparation), Factor IX (Haemophilia B), Factor VIIa(Haemorrhage in patients with haemophilia A or B and inhibitors tofactor VIII or factor IX), Factor VIII (Haemophilia A), Lepirudin(Heparin-induced thrombocytopaenia), Protein C concentrate (Venousthrombosis, Purpura fulminans), Reteplase (deletion mutein of tPA)(Management of acute myocardial infarction, improvement of ventricularfunction), Streptokinase (Acute evolving transmural myocardialinfarction, pulmonary embolism, deep vein thrombosis, arterialthrombosis or embolism, occlusion of arteriovenous cannula),Tenecteplase (Acute myocardial infarction), Urokinase (Pulmonaryembolism), Angiostatin (Cancer), Anti-CD22 immunotoxin (Relapsed CD33+acute myeloid leukaemia), Denileukin diftitox (Cutaneous T-cell lymphoma(CTCL)), Immunocyanin (bladder and prostate cancer), MPS(Metallopanstimulin) (Cancer), Aflibercept (Non-small cell lung cancer(NSCLC), metastatic colorectal cancer (mCRC), hormone-refractorymetastatic prostate cancer, wet macular degeneration), Endostatin(Cancer, inflammatory diseases like rheumatoid arthritis as well asCrohn's disease, diabetic retinopathy, psoriasis, and endometriosis),Collagenase (Debridement of chronic dermal ulcers and severely burnedareas, Dupuytren's contracture, Peyronie's disease), Humandeoxy-ribonuclease I, dornase (Cystic fibrosis; decreases respiratorytract infections in selected patients with FVC greater than 40% ofpredicted), Hyaluronidase (Used as an adjuvant to increase theabsorption and dispersion of injected drugs, particularly anaestheticsin ophthalmic surgery and certain imaging agents), Papain (Debridementof necrotic tissue or liquefication of slough in acute and chroniclesions, such as pressure ulcers, varicose and diabetic ulcers, burns,postoperative wounds, pilonidal cyst wounds, carbuncles, and otherwounds), L-Asparaginase (Acute lymphocytic leukaemia, which requiresexogenous asparagine for proliferation), Peg-asparaginase (Acutelymphocytic leukaemia, which requires exogenous asparagine forproliferation), Rasburicase (Paediatric patients with leukaemia,lymphoma, and solid tumours who are undergoing anticancer therapy thatmay cause tumour lysis syndrome), Human chorionic gonadotropin (HCG)(Assisted reproduction), Human follicle-stimulating hormone (FSH)(Assisted reproduction), Lutropin-alpha (Infertility with luteinizinghormone deficiency), Prolactin (Hypoprolactinemia, serum prolactindeficiency, ovarian dysfunction in women, anxiety, arteriogenic erectiledysfunction, premature ejaculation, oligozoospermia, asthenospermia,hypofunction of seminal vesicles, hypoandrogenism in men),alpha-1-Proteinase inhibitor (Congenital antitrypsin deficiency),Lactase (Gas, bloating, cramps and diarrhoea due to inability to digestlactose), Pancreatic enzymes (lipase, amylase, protease) (Cysticfibrosis, chronic pancreatitis, pancreatic insufficiency, post-BillrothII gastric bypass surgery, pancreatic duct obstruction, steatorrhoea,poor digestion, gas, bloating), Adenosine deaminase (pegademase bovine,PEG-ADA) (Severe combined immunodeficiency disease due to adenosinedeaminase deficiency), Abatacept (Rheumatoid arthritis (especially whenrefractory to TNFa inhibition)), Alefacept (Plaque Psoriasis), Anakinra(Rheumatoid arthritis), Etanercept (Rheumatoid arthritis,polyarticular-course juvenile rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, plaque psoriasis, ankylosing spondylitis),Interleukin-1 (IL-1) receptor antagonist, Anakinra (inflammation andcartilage degradation associated with rheumatoid arthritis), Thymulin(neurodegenerative diseases, rheumatism, anorexia nervosa), TNF-alphaantagonist (autoimmune disorders such as rheumatoid arthritis,ankylosing spondylitis, Crohn's disease, psoriasis, hidradenitissuppurativa, refractory asthma), Enfuvirtide (HIV-1 infection), andThymosin α1 (Hepatitis B and C).

(in brackets is the particular disease, for which the therapeuticprotein is used in the treatment)

Furthermore, adjuvant or immunostimulating proteins are also encompassedin the term therapeutic proteins. Adjuvant or immunostimulating proteinsmay be used in this context to induce, alter or improve an immuneresponse in an individual to treat a particular disease or to amelioratethe condition of the individual.

In this context, adjuvant proteins may be selected from mammalian, inparticular human adjuvant proteins, which typically comprise any humanprotein or peptide, which is capable of eliciting an innate immuneresponse (in a mammal), e.g. as a reaction of the binding of anexogenous TLR ligand to a TLR. More preferably, human adjuvant proteinsare selected from the group consisting of proteins, which are componentsand ligands of the signalling networks of the pattern recognitionreceptors including TLR, NLR and RLH, including TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; NOD1, NOD2, NOD3, NOD4,NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP6, NALP7, NALP7,NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14,1 IPAF, NAIP,CIITA, RIG-I, MDA5 and LGP2, the signal transducers of TLR signalingincluding adaptor proteins including e.g. Trif and Cardif; components ofthe Small-GTPases signalling (RhoA, Ras, Rac1, Cdc42, Rab etc.),components of the PIP signalling (PI3K, Src-Kinases, etc.), componentsof the MyD88-dependent signalling (MyD88, IRAK1, IRAK2, IRAK4, TIRAP,TRAF6 etc.), components of the MyD88-independent signalling (TICAM1,TICAM2, TRAF6, TBK1, IRF3, TAK1, IRAK1 etc.); the activated kinasesincluding e.g. Akt, MEKK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1, ERK2,GSK3, PKC kinases, PKD kinases, GSK3 kinases, JNK, p38MAPK, TAK1, IKK,and TAK1; the activated transcription factors including e.g. NF-κB,c-Fos, c-Jun, c-Myc, CREB, AP-1, Elk-1, ATF2, IRF-3, IRF-7.

Mammalian, in particular human adjuvant proteins may furthermore beselected from the group consisting of heat shock proteins, such asHSP10, HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen, TypIIIrepeat extra domain A of fibronectin; or components of the complementsystem including Clq, MBL, Clr, Cls, C2b, Bb, D, MASP-1, MASP-2, C4b,C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR,CIINH, C4 bp, MCP, DAF, H, I, P and CD59, or induced target genesincluding e.g. Beta-Defensin, cell surface proteins; or human adjuvantproteins including trif, flt-3 ligand, Gp96 or fibronectin, etc., or anyspecies homolog of any of the above human adjuvant proteins.

Mammalian, in particular human adjuvant proteins may furthermorecomprise cytokines which induce or enhance an innate immune response,including IL-1 alpha, IL1 beta, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12,IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, TNFalpha, IFNalpha,IFNbeta, IFNgamma, GM-CSF, G-CSF, M-CSF; chemokines including IL-8,IP-10, MCP-1, MIP-lalpha, RANTES, Eotaxin, CCL21; cytokines which arereleased from macrophages, including IL-1, IL-6, IL-8, IL-12 andTNF-alpha; as well as IL-IR1 and IL-1 alpha.

Therapeutic proteins for the treatment of blood disorders, diseases ofthe circulatory system, diseases of the respiratory system, cancer ortumour diseases, infectious diseases or immunedeficiencies or adjuvantproteins are typically proteins of mammalian origin, preferably of humanorigin, depending on which animal shall be treated. A human subject, forexample, is preferably treated by a therapeutic protein of human origin.

Pathogenic adjuvant proteins, typically comprise a pathogenic adjuvantprotein, which is capable of eliciting an innate immune response (in amammal), more preferably selected from pathogenic adjuvant proteinsderived from bacteria, protozoa, viruses, or fungi, etc., e.g.,bacterial (adjuvant) proteins, protozoan (adjuvant) proteins (e.g.profilin—like protein of Toxoplasma gondii), viral (adjuvant) proteins,or fungal (adjuvant) proteins, etc.

Particularly, bacterial (adjuvant) proteins may be selected from thegroup consisting of bacterial heat shock proteins or chaperons,including Hsp60, Hsp70, Hsp90, Hsp100; OmpA (Outer membrane protein)from gram-negative bacteria; bacterial porins, including OmpF; bacterialtoxins, including pertussis toxin (PT) from Bordetella pertussis,pertussis adenylate cyclase toxin CyaA and CyaC from Bordetellapertussis, PT-9K/129G mutant from pertussis toxin, pertussis adenylatecyclase toxin CyaA and CyaC from Bordetella pertussis, tetanus toxin,cholera toxin (CT), cholera toxin B-subunit, CTK63 mutant from choleratoxin, CTE112K mutant from CT, Escherichia coli heat-labile enterotoxin(LT), B subunit from heat-labile enterotoxin (LTB) Escherichia coliheat-labile enterotoxin mutants with reduced toxicity, including LTK63,LTR72; phenol-soluble modulin; neutrophil-activating protein (HP-NAP)from Helicobacter pylori; Surfactant protein D; Outer surface protein Alipoprotein from Borrelia burgdorferi, Ag38 (38 kDa antigen) fromMycobacterium tuberculosis; proteins from bacterial fimbriae;Enterotoxin CT of Vibrio cholerae, Pilin from pili from gram negativebacteria, and Surfactant protein A; etc., or any species homolog of anyof the above bacterial (adjuvant) proteins.

Bacterial (adjuvant) proteins may also comprise bacterial flagellins. Inthe context of the present invention, bacterial flagellins may beselected from flagellins from organisms including, without being limitedthereto, Agrobacterium, Aquifex, Azospirillum, Bacillus, Bartonella,Bordetella, Borrelia, Burkholderia, Campylobacter, Caulobacte,Clostridium, Escherichia, Helicobacter, Lachnospiraceae, Legionella,Listeria, Proteus, Pseudomonas, Rhizobium, Rhodobacter, Roseburia,Salmonella, Serpulina, Serratia, Shigella, Treponema, Vibrio, Wolinella,Yersinia, more preferably from flagellins from the species including,without being limited thereto, Agrobacterium tumefaciens, Aquifexpyrophilus, Azospirillum brasilense, Bacillus subtilis, Bacillusthuringiensis, Bartonella bacilliformis, Bordetella bronchiseptica,Borrelia burgdorferi, Burkholderia cepacia, Campylobacter jejuni,Caulobacter crescentus, Clostridium botulinum strain Bennett clone 1,Escherichia coli, Helicobacter pylori, Lachnospiraceae bacterium,Legionella pneumophila, Listeria monocytogenes, Proteus mirabilis,Pseudomonas aeroguinosa, Pseudomonas syringae, Rhizobium meliloti,Rhodobacter sphaeroides, Roseburia cecicola, Roseburis hominis,Salmonella typhimurium, Salmonella bongori, Salmonella typhi, Salmonellaenteritidis, Serpulina hyodysenteriae, Serratia marcescens, Shigellaboydii, Treponema phagedenis, Vibrio alginolyticus, Vibrio cholerae,Vibrio parahaemolyticus, Wolinella succinogenes and Yersiniaenterocolitica.

Protozoan (adjuvant) proteins are a further example of pathogenicadjuvant proteins. Protozoan (adjuvant) proteins may be selected in thiscontext from any protozoan protein showing adjuvant properties, morepreferably, from the group consisting of, without being limited thereto,Tc52 from Trypanosoma cruzi, PFTG from Trypanosoma gondii, Protozoanheat shock proteins, LeIF from Leishmania spp., profiling-like proteinfrom Toxoplasma gondii, etc.

Viral (adjuvant) proteins are another example of pathogenic adjuvantproteins. In this context, viral (adjuvant) proteins may be selectedfrom any viral protein showing adjuvant properties, more preferably,from the group consisting of, without being limited thereto, RespiratorySyncytial Virus fusion glycoprotein (F-protein), envelope protein fromMMT virus, mouse leukemia virus protein, Hemagglutinin protein ofwild-type measles virus, etc.

Fungal (adjuvant) proteins are even a further example of pathogenicadjuvant proteins. In the context of the present invention, fungal(adjuvant) proteins may be selected from any fungal protein showingadjuvant properties, more preferably, from the group consisting of,fungal immunomodulatory protein (FIP; LZ-8), etc.

Finally, adjuvant proteins may furthermore be selected from the groupconsisting of, Keyhole limpet hemocyanin (KLH), OspA, etc.

In a further embodiment, therapeutic proteins may be used for hormonereplacement therapy, particularly for the therapy of women in themenopause. These therapeutic proteins are preferably selected fromoestrogens, progesterone or progestins, and sometimes testosterone.

Furthermore, therapeutic proteins may be used for reprogramming ofsomatic cells into pluri- or omnipotent stem cells. For this purpose,several factors are described, particularly Oct-3/4, Sox gene family(Sox1, Sox2, Sox3, and Sox15), Klf family (Klf1, Klf2, Klf4, and Klf5),Myc family (c-myc, L-myc, and N-myc), Nanog, and LIN28.

As mentioned above, also therapeutic antibodies are defined herein astherapeutic proteins. These therapeutic antibodies are preferablyselected from antibodies, which are used inter alia for the treatment ofcancer or tumour diseases, e.g. 131I-tositumomab (Follicular lymphoma, Bcell lymphomas, leukemias), 3F8 (Neuroblastoma), 8H9, Abagovomab(Ovarian cancer), Adecatumumab (Prostate and breast cancer), Afutuzumab(Lymphoma), Alacizumab pegol, Alemtuzumab (B-cell chronic lymphocyticleukaemia, T-cell-Lymphoma), Amatuximab, AME-133v (Follicular lymphoma,cancer), AMG 102 (Advanced Renal Cell Carcinoma), Anatumomab mafenatox(Non-small cell lung carcinoma), Apolizumab (Solid Tumors, Leukemia,Non-Hodgkin-Lymphoma, Lymphoma), Bavituximab (Cancer, viral infections),Bectumomab (Non-Hodgkin's lymphoma), Belimumab (Non-Hodgkin lymphoma),Bevacizumab (Colon Cancer, Breast Cancer, Brain and Central NervousSystem Tumors, Lung Cancer, Hepatocellular Carcinoma, Kidney Cancer,Breast Cancer, Pancreatic Cancer, Bladder Cancer, Sarcoma, Melanoma,Esophageal Cancer; Stomach Cancer, Metastatic Renal Cell Carcinoma;Kidney Cancer, Glioblastoma, Liver Cancer, Proliferative DiabeticRetinopathy, Macular Degeneration), Bivatuzumab mertansine (Squamouscell carcinoma), Blinatumomab, Brentuximab vedotin (Hematologiccancers), Cantuzumab (Colon Cancer, Gastric Cancer, Pancreatic Cancer,NSCLC), Cantuzumab mertansine (Colorectal cancer), Cantuzumab ravtansine(Cancers), Capromab pendetide (Prostate cancer), Carlumab, Catumaxomab(Ovarian Cancer, Fallopian Tube Neoplasms, Peritoneal Neoplasms),Cetuximab (Metastatic colorectal cancer and head and neck cancer),Citatuzumab bogatox (Ovarian cancer and other solid tumors), Cixutumumab(Solid tumors), Clivatuzumab tetraxetan (Pancreatic cancer), CNTO 328(B-Cell Non-Hodgkin's Lymphoma, Multiple Myeloma, Castleman's Disease,ovarian cancer), CNTO 95 (Melanoma), Conatumumab, Dacetuzumab(Hematologic cancers), Dalotuzumab, Denosumab (Myeloma, Giant Cell Tumorof Bone, Breast Cancer, Prostate Cancer, Osteoporosis), Detumomab(Lymphoma), Drozitumab, Ecromeximab (Malignant melanoma), Edrecolomab(Colorectal carcinoma), Elotuzumab (Multiple myeloma), Elsilimomab,Enavatuzumab, Ensituximab, Epratuzumab (Autoimmune diseases, SystemicLupus Erythematosus, Non-Hodgkin-Lymphoma, Leukemia), Ertumaxomab(Breast cancer), Ertumaxomab (Breast Cancer), Etaracizumab (Melanoma,prostate cancer, ovarian cancer), Farletuzumab (Ovarian cancer), FBTA05(Chronic lymphocytic leukaemia), Ficlatuzumab (Cancer), Figitumumab(Adrenocortical carcinoma, non-small cell lung carcinoma), Flanvotumab(Melanoma), Galiximab (B-cell lymphoma), Galiximab(Non-Hodgkin-Lymphoma), Ganitumab, GC1008 (Advanced Renal CellCarcinoma; Malignant Melanoma, Pulmonary Fibrosis), Gemtuzumab(Leukemia), Gemtuzumab ozogamicin (Acute myelogenous leukemia),Girentuximab (Clear cell renal cell carcinoma), Glembatumumab vedotin(Melanoma, breast cancer), GS6624 (Idiopathic pulmonary fibrosis andsolid tumors), HuC242-DM4 (Colon Cancer, Gastric Cancer, PancreaticCancer), HuHMFG1 (Breast Cancer), HuN901-DM1 (Myeloma), Ibritumomab(Relapsed or refractory low-grade, follicular, or transformed B-cellnon-Hodgkin's lymphoma (NHL)), Icrucumab, ID09C3 (Non-Hodgkin-Lymphoma),Indatuximab ravtansine, Inotuzumab ozogamicin, Intetumumab (Solid tumors(Prostate cancer, melanoma)), Ipilimumab (Sarcoma, Melanoma, Lungcancer, Ovarian Cancer leucemia, Lymphoma, Brain and Central NervousSystem Tumors, Testicular Cancer, Prostate Cancer, Pancreatic Cancer,Breast Cancer), Iratumumab (Hodgkin's lymphoma), Labetuzumab (Colorectalcancer), Lexatumumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab(Multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma),Lumiliximab (Chronic lymphocytic leukemia), Mapatumumab (Colon Cancer,Myeloma), Matuzumab (Lung Cancer, Cervical Cancer, Esophageal Cancer),MDX-060 (Hodgkin-Lymphoma, Lymphoma), MEDI 522 (Solid Tumors, Leukemia,Lymphoma, Small Intestine Cancer, Melanoma), Mitumomab (Small cell lungcarcinoma), Mogamulizumab, MORab-003 (Ovarian Cancer, Fallopian TubeCancer, Peritoneal Cancer), MORab-009 (Pancreatic Cancer, Mesothelioma,Ovarian Cancer, Non-Small Cell Lung Cancer, Fallopian Tube Cancer,Peritoneal Cavity Cancer), Moxetumomab pasudotox, MT103(Non-Hodgkin-Lymphoma), Nacolomab tafenatox (Colorectal cancer),Naptumomab estafenatox (Non-small cell lung carcinoma, renal cellcarcinoma), Narnatumab, Necitumumab (Non-small cell lung carcinoma),Nimotuzumab (Squamous cell carcinoma, head and neck cancer,nasopharyngeal cancer, glioma), Nimotuzumab (Squamous cell carcinomas,Glioma, Solid Tumors, Lung Cancer), Olaratumab, Onartuzumab (Cancer),Oportuzumab monatox, Oregovomab (Ovarian cancer), Oregovomab (OvarianCancer, Fallopian Tube Cancer, Peritoneal Cavity Cancer), PAM4(Pancreatic Cancer), Panitumumab (Colon Cancer, Lung Cancer, BreastCancer; Bladder Cancer; Ovarian Cancer), Patritumab, Pemtumomab,Pertuzumab (Breast Cancer, Ovarian Cancer, Lung Cancer, ProstateCancer), Pritumumab (Brain cancer), Racotumomab, Radretumab, Ramucirumab(Solid tumors), Rilotumumab (Solid tumors), Rituximab (Urticaria,Rheumatoid Arthritis, Ulcerative Colitis, Chronic Focal Encephalitis,Non-Hodgkin-Lymphoma, Lymphoma, Chronic Lymphocytic Leukemia),Robatumumab, Samalizumab, SGN-30 (Hodgkin-Lymphoma, Lymphoma), SGN-40(Non-Hodgkin-Lymphoma, Myeloma, Leukemia, Chronic Lymphocytic Leukemia),Sibrotuzumab, Siltuximab, Tabalumab (B-cell cancers), Tacatuzumabtetraxetan, Taplitumomab paptox, Tenatumomab, Teprotumumab (Hematologictumors), TGN1412 (Chronic lymphocytic leukemia, rheumatoid arthritis),Ticilimumab (=tremelimumab), Tigatuzumab, TNX-650 (Hodgkin's lymphoma),Tositumomab (Follicular lymphoma, B cell lymphomas, Leukemias, Myeloma),Trastuzumab (Breast Cancer, Endometrial Cancer, Solid Tumors), TRBS07(Melanoma), Tremelimumab, TRU-016 (Chronic lymphocytic leukemia),TRU-016 (Non-Hodgkin lymphoma), Tucotuzumab celmoleukin, Ublituximab,Urelumab, Veltuzumab (Non-Hodgkin's lymphoma), Veltuzumab (IMMU-106)(Non-Hodgkin's lymphoma), Volociximab (Renal Cell Carcinoma, PancreaticCancer, Melanoma), Votumumab (Colorectal tumors), WX-G250 (Renal CellCarcinoma), Zalutumumab (Head and Neck Cancer, Squamous Cell Cancer),and Zanolimumab (T-Cell-Lymphoma);

antibodies, which are used inter alia for the treatment of immunedisorders, e.g. Efalizumab (Psoriasis), Epratuzumab (Autoimmunediseases, Systemic Lupus Erythematosus, Non-Hodgkin-Lymphoma, Leukemia),Etrolizumab (inflammatory bowel disease), Fontolizumab (Crohn'sdisease), Ixekizumab (autoimmune diseases), Mepolizumab(Hypereosinophilie-Syndrom, Asthma, Eosinophilic Gastroenteritis,Churg-Strauss Syndrome, Eosinophilic Esophagitis), Milatuzumab (multiplemyeloma and other hematological malignancies), Pooled immunoglobulins(Primary immunodeficiencies), Priliximab (Crohn's disease, multiplesclerosis), Rituximab (Urticaria, Rheumatoid Arthritis, UlcerativeColitis, Chronic Focal Encephalitis, Non-Hodgkin-Lymphoma, Lymphoma,Chronic Lymphocytic Leukemia), Rontalizumab (systemic lupuserythematosus), Ruplizumab (rheumatic diseases), Sarilumab (rheumatoidarthritis, ankylosing spondylitis), Vedolizumab (Crohn's disease,ulcerative colitis), Visilizumab (Crohn's disease, ulcerative colitis),Reslizumab (inflammations of the airways, skin and gastrointestinaltract), Adalimumab (Rheumatoid arthritis, Crohn's disease, Ankylosingspondylitis, Psoriatic arthritis), Aselizumab (severely injuredpatients), Atinumab (treatment of neurologic systems), Atlizumab(rheumatoid arthritis, systemic juvenile idiopathic arthritis),Bertilimumab (severe allergic disorders), Besilesomab (inflammatorylesions and metastases), BMS-945429, ALD518 (cancer and rheumatoidarthritis), Briakinumab (psoriasis, rheumatoid arthritis, inflammatorybowel diseases, multiple sclerosis), Brodalumab (inflammatory diseases),Canakinumab (rheumatoid arthritis), Canakinumab (cryopyrin-associatedperiodic syndromes (CAPS), rheumatoid arthritis, chronic obstructivepulmonary disease), Certolizumab pegol (Crohn's disease), Erlizumab(heart attack, stroke, traumatic shock), Fezakinumab (rheumatoidarthritis, psoriasis), Golimumab (rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis), Gomiliximab (allergic asthma),Infliximab (Rheumatoid arthritis, Crohn's disease, ankylosingspondylitis, psoriatic arthritis, plaque psoriasis, Morbus Bechterew,Colitis ulcerosa), Mavrilimumab (rheumatoid arthritis), Natalizumab(Multiple sclerosis), Ocrelizumab (multiple sclerosis, rheumatoidarthritis, lupus erythematosus, hematological cancer), Odulimomab(prevention of organ transplant rejections, immunological diseases),Ofatumumab (Chronic lymphocytic leukemia, follicular non-Hodgkin'slymphoma, B cell lymphoma, rheumatoid arthritis, relapsing remittingmultiple sclerosis, Lymphoma, B-Cell Chronic Lymphocytic Leukemia),Ozoralizumab (inflammation), Pexelizumab (reduction of side effects ofcardiac surgery), Rovelizumab (haemorrhagic shock), SBI-087 (Rheumatoidarthritis), SBI-087 (Systemic lupus erythematosus), Secukinumab(uveitis, rheumatoid arthritis psoriasis), Sirukumab (rheumatoidarthritis), Talizumab (allergic reaction), Tocilizumab (rheumatoidarthritis, systemic juvenile idiopathic arthritis, Castleman's disease),Toralizumab (rheumatoid arthritis, lupus nephritis), TRU-015 (Rheumatoidarthritis), TRU-016 (Autoimmune disease and inflammation), Ustekinumab(multiple sclerosis, psoriasis, psoriatic arthritis), Ustekinumab(IL-12/IL-23 blocker) (Plaque-Psoriasis, psoriatic arthritis, multiplesclerosis, sarcoidosis, the latter versus), Vepalimomab (inflammation),Zolimomab aritox (systemic lupus erythematosus, graft-versus-hostdisease), Sifalimumab (SLE, dermatomyositis, polymyositis), Lumiliximab(Allergies), and Rho(D) Immune Globulin (Rhesus disease); or areselected from antibodies used for the treatment of infectious diseases,e.g. Afelimomab (sepsis), CR6261 (infectious disease/influenza A),Edobacomab (sepsis caused by gram-negative bacteria), Efungumab(invasive Candida infection), Exbivirumab (hepatitis B), Felvizumab(respiratory syncytial virus infection), Foravirumab (rabies(prophylaxis)), Ibalizumab (HIV infection), Libivirumab (hepatitis B),Motavizumab (respiratory syncytial virus (prevention)), Nebacumab(sepsis), Tuvirumab (chronic hepatitis B), Urtoxazumab (diarrhoea causedby E. coli), Bavituximab (diverse viral infections), Pagibaximab (sepsis(e.g. Staphylococcus)), Palivizumab (prevention of respiratory syncytialvirus infection in high-risk paediatric patients), Panobacumab(Pseudomonas aeruginosa infection), PRO 140 (HIV infection), Rafivirumab(rabies (prophylaxis)), Raxibacumab (anthrax (prophylaxis andtreatment)), Regavirumab (cytomegalovirus infection), Sevirumab(cytomegalovirus infection), Suvizumab (viral infections), andTefibazumab (Staphylococcus aureus infection);

antibodies, which are used inter alia for the treatment of blooddisorders, e.g. Abciximab (percutaneous coronary intervention),Atorolimumab (hemolytic disease of the newborn), Eculizumab (Paroxysmalnocturnal haemoglobinuria), Mepolizumab (Hypereosinophilie-Syndrom,Asthma, Eosinophilic Gastroenteritis, Churg-Strauss Syndrome,Eosinophilic Esophagitis), and Milatuzumab (multiple myeloma and otherhematological malignancies);antibodies, which are used inter alia for immunoregulation, e.g.Antithymocyte globulin (Acute kidney transplant rejection, aplasticanaemia), Basiliximab (Prophylaxis against allograft rejection in renaltransplant patients receiving an immunosuppressive regimen includingcyclosporine and corticosteroids), Cedelizumab (prevention of organtransplant rejections, treatment of autoimmune diseases), Daclizumab(Prophylaxis against acute allograft rejection in patients receivingrenal transplants, Multiple Sclerosis), Gavilimomab (graft versus hostdisease), Inolimomab (graft versus host disease), Muromonab-CD3(prevention of organ transplant rejections), Muromonab-CD3 (Acute renalallograft rejection or steroid-resistant cardiac or hepatic allograftrejection), Odulimomab (prevention of organ transplant rejections,immunological diseases), and Siplizumab (psoriasis, graft-versus-hostdisease (prevention));antibodies used for the treatment of diabetes, e.g. Gevokizumab(diabetes), Otelixizumab (diabetes mellitus type 1), and Teplizumab(diabetes mellitus type 1);antibodies, which are used for the treatment of the Alzheimer's disease,e.g. Bapineuzumab, Crenezumab, Gantenerumab, Ponezumab, R1450, andSolanezumab;antibodies, which are used for the treatment of asthma, e.g.Benralizumab, Enokizumab, Keliximab, Lebrikizumab, Omalizumab, Oxelumab,Pascolizumab, and Tralokinumab;and antibodies, which are used for the treatment of diverse disorders,e.g. Blosozumab (osteoporosis), CaroRx (Tooth decay), Fresolimumab(idiopathic pulmonary fibrosis, focal segmental glomerulosclerosis,cancer), Fulranumab (pain), Romosozumab (osteoporosis), Stamulumab(muscular dystrophy), Tanezumab (pain), and Ranibizumab (Neovascularage-related macular degeneration).

The coding region of the modified RNA according to the present inventionmay occur as a mono-, di-, or even multicistronic RNA, i.e. an RNA,which carries the coding sequences of one, two or more proteins orpeptides. Such coding sequences in di-, or even multicistronic RNA's maybe separated by at least one internal ribosome entry site (IRES)sequence, e.g. as described herein or by signal peptides which inducethe cleavage of the resulting polypeptide, which comprises severalproteins or peptides.

Pharmaceutical Composition:

Additionally, according to another aspect, the present invention alsorelates to the use of the modified RNA as defined herein or of acomposition comprising a plurality of modifed RNA molecules as definedherein for the preparation of a pharmaceutical composition forincreasing the expression of an encoded peptide or protein, particularlyfor use in gene therapy or genetic vaccination, e.g. for treating adisease, preferably as defined herein, e.g. applying or administeringthe modifed RNA as defined herein or of a composition comprising aplurality of modified RNA molecules as defined herein to a cell (e.g. anexpression host cell or a somatic cell), a tissue or an organism,preferably in naked form or complexed form or as a pharmaceuticalcomposition as described herein by jet injection.

Accordingly, in a particular preferred aspect, the present inventionalso provides a pharmaceutical composition, comprising a modified RNA asdefined herein or a composition comprising a plurality of modified RNA'sas defined herein and optionally a pharmaceutically acceptable carrierand/or vehicle for administration by jet injection.

As a first ingredient, the pharmaceutical composition comprises at leastone modified nucleic acid as defined herein.

As a second ingredient the pharmaceutical composition may optionallycomprise at least one additional pharmaceutically active component. Apharmaceutically active component in this connection is a compound thathas a therapeutic effect to heal, ameliorate or prevent a particularindication or disease as mentioned herein. Such compounds include,without implying any limitation, peptides or proteins, preferably asdefined herein, nucleic acids, preferably as defined herein,(therapeutically active) low molecular weight organic or inorganiccompounds (molecular weight less than 5000, preferably less than 1000),sugars, antigens or antibodies, preferably as defined herein,therapeutic agents already known in the prior art, antigenic cells,antigenic cellular fragments, cellular fractions; cell wall components(e.g. polysaccharides), modified, attenuated or de-activated (e.g.chemically or by irradiation) pathogens (virus, bacteria etc.),adjuvants, preferably as defined herein, etc.

Furthermore, the pharmaceutical composition may comprise apharmaceutically acceptable carrier and/or vehicle. In the context ofthe present invention, a pharmaceutically acceptable carrier typicallyincludes the liquid or non-liquid basis of the inventive pharmaceuticalcomposition. The carrier will typically be pyrogen-free water, isotonicsaline or buffered (aqueous) solutions, e.g phosphate, citrate etc.buffered solutions. The injection buffer may be hypertonic, isotonic orhypotonic with reference to the specific reference medium, i.e. thebuffer may have a higher, identical or lower salt content with referenceto the specific reference medium, wherein preferably such concentrationsof the afore mentioned salts may be used, which do not lead to damage ofcells due to osmosis or other concentration effects. Reference media aree.g. liquids occurring in “in vivo” methods, such as blood, lymph,cytosolic liquids, or other body liquids, or e.g. liquids, which may beused as reference media in “in vitro” methods, such as common buffers orliquids. Such common buffers or liquids are known to a skilled person.Ringer-Lactate solution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well for the pharmaceuticalcomposition, which are suitable for administration to a patient to betreated. The term “compatible” as used here means that theseconstituents of the pharmaceutical composition are capable of beingmixed with the modified RNA as defined herein in such a manner that nointeraction occurs which would substantially reduce the pharmaceuticaleffectiveness of the pharmaceutical composition under typical useconditions.

Complexation:

Furthermore, the pharmaceutical composition may comprise a carrier forthe modified RNA. Such a carrier may be suitable for mediatingdissolution in physiological acceptable liquids, transport and cellularuptake of the pharmaceutically active modified RNA molecule.Accordingly, such a carrier may be a component, which may be suitablefor depot and delivery of a modified RNA according to the invention.Such components may be, for example, cationic or polycationic carriersor compounds, which may serve as transfection or complexation agent.

Particularly preferred transfection or complexation agents in thiscontext are cationic or polycationic compounds, including protamine,nucleoline, spermine or spermidine, or other cationic peptides orproteins, such as poly-L-lysine (PLL), poly-arginine, basicpolypeptides, cell penetrating peptides (CPPs), including HIV-bindingpeptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA orprotein transduction domains (PTDs), PpT620, proline-rich peptides,arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1,L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, plsl, FGF,Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC,hCT-derived peptides, SAP, or histones.

Furthermore, such cationic or polycationic compounds or carriers may becationic or polycationic peptides or proteins, which preferably compriseor are additionally modified to comprise at least one —SH moiety.Preferably, a cationic or polycationic carrier is selected from cationicpeptides having the following sum formula (III):

{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)};  formula (III)

wherein l+m+n+o+x=3-100, and 1, m, n or o independently of each other isany number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80,81-90 and 91-100 provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide; and Xaa is any amino acidselected from native (=naturally occurring) or non-native amino acidsexcept of Arg, Lys, His or Orn; and x is any number selected from 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, provided, that theoverall content of Xaa does not exceed 90% of all amino acids of theoligopeptide. Any of amino acids Arg, Lys, His, Ornm and Xaa may bepositioned at any place of the peptide. In this context cationicpeptides or proteins in the range of 7-30 amino acids are particularpreferred.

Further, the cationic or polycationic peptide or protein, when definedaccording to formula {(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (III)) as shown aboveand which comprise or are additionally modified to comprise at least one—SH moeity, may be, without being restricted thereto, selected fromsubformula (Ia):

{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa′)_(x)(Cys)_(y)}  subformula(IIIa)

wherein (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o); and x are as definedherein, Xaa′ is any amino acid selected from native (=naturallyoccurring) or non-native amino acids except of Arg, Lys, His, Orn or Cysand y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70,71-80 and 81-90, provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide. Further, the cationic orpolycationic peptide may be selected from subformula (IIIb):

Cys₁{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}Cys₂  subformula(IIIb)

wherein empirical formula{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (IV)) is asdefined herein and forms a core of an amino acid sequence according to(semiempirical) formula (IV) and wherein Cys₁ and Cys₂ are Cysteinesproximal to, or terminal to(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x).

Further preferred cationic or polycationic compounds, which can be usedas transfection or complexation agent may include cationicpolysaccharides, for example chitosan, polybrene, cationic polymers,e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium, CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g polyethyleneglycole); etc.

In this context, it is particularly preferred that the modified RNAmolecule is complexed at least partially with a cationic or polycationiccompound, preferably cationic proteins or peptides. Partially means thatonly a part of the modified RNA molecule is complexed with a cationic orpolycationic compound and that the rest of the modified RNA molecule isin uncomplexed form (“free”). Preferably the ratio of complexed modifiedRNA to free modified RNA is selected from a range of about 5:1 (w/w) toabout 1:10 (w/w), more preferably from a range of about 4:1 (w/w) toabout 1:8 (w/w), even more preferably from a range of about 3:1 (w/w) toabout 1:5 (w/w) or 1:3 (w/w), and most preferably the ratio of complexednucleic acid to free nucleic acid is selected from a ratio of about 1:1(w/w).

According to a specific embodiment, the pharmaceutical composition maycomprise an adjuvant. In this context, an adjuvant may be understood asany compound, which is suitable to initiate or increase an immuneresponse of the innate immune system, i.e. a non-specific immuneresponse. With other words, when administered, the inventivepharmaceutical composition preferably elicits an innate immune responsedue to the adjuvant, optionally contained therein. Preferably, such anadjuvant may be selected from an adjuvant known to a skilled person andsuitable for the present case, i.e. supporting the induction of aninnate immune response in a mammal, e.g. an adjuvant protein as definedabove or an adjuvant as defined in the following.

Particularly preferred as adjuvants suitable for depot and delivery arecationic or polycationic compounds as defined above for the modified RNAas vehicle, transfection or complexation agent.

Further additives which may be included in the inventive pharmaceuticalcomposition are emulsifiers, such as, for example, Tween®; wettingagents, such as, for example, sodium lauryl sulfate; colouring agents;taste-imparting agents, pharmaceutical carriers; tablet-forming agents;stabilizers; antioxidants; preservatives.

The pharmaceutical composition can also additionally contain any furthercompound, which is known to be immunostimulating due to its bindingaffinity (as ligands) to human Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its bindingaffinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

The pharmaceutical composition may preferably be administeredsubcutaneously, intramuscularly or intradermally by jet injection.Sterile injectable forms of the pharmaceutical compositions may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents.

The pharmaceutical composition typically comprises a “safe and effectiveamount” of the components of the pharmaceutical composition,particularly of the modified RNA as defined herein. As used herein, a“safe and effective amount” means an amount of the modified RNA asdefined herein as such that is sufficient to significantly induce apositive modification of a disease or disorder as defined herein. At thesame time, however, a “safe and effective amount” is small enough toavoid serious side-effects and to permit a sensible relationship betweenadvantage and risk. The determination of these limits typically lieswithin the scope of sensible medical judgment.

The present invention furthermore provides several applications and usesof the modified RNA as defined herein, of the composition comprising aplurality of modifed RNA molecules as defined herein, of thepharmaceutical composition, comprising the modified RNA as definedherein or of kits comprising same.

According to one specific aspect, the present invention is directed tothe first medical use of the inventive modified RNA as defined herein orof the inventive composition comprising a plurality of inventive RNAmolecules as defined herein as a medicament, particularly in genetherapy or genetic vaccination, preferably for the treatment of diseasesas defined herein. According to another aspect, the present invention isdirected to the second medical use of the inventive modified RNA asdefined herein or of the inventive composition comprising a plurality ofinventive modified RNA molecules as defined herein, for the treatment ofdiseases as defined herein, preferably to the use of the inventivemodified RNA as defined herein, of the inventive composition comprisinga plurality of inventive modified RNA molecules as defined herein, of apharmaceutical composition comprising same or of kits comprising samefor the preparation of a medicament for the prophylaxis, treatmentand/or amelioration of diseases as defined herein. Preferably, thepharmaceutical composition is used or to be administered to a patient inneed thereof for this purpose.

Preferably, diseases as mentioned herein are preferably selected frominfectious diseases, neoplasms (e.g. cancer or tumour diseases),diseases of the blood and blood-forming organs, endocrine, nutritionaland metabolic diseases, diseases of the nervous system, diseases of thecirculatory system, diseases of the respiratory system, diseases of thedigestive system, diseases of the skin and subcutaneous tissue, diseasesof the musculoskeletal system and connective tissue, and diseases of thegenitourinary system.

Diseases: Infectious Diseases:

Preferably, infectious diseases as mentioned herein are preferablyselected from viral, bacterial, protozoological and prion infectiousdiseases. Such infectious diseases are typically selected from the listconsisting of Acinetobacter infections, African sleeping sickness(African trypanosomiasis), AIDS (Acquired immunodeficiency syndrome),Amoebiasis, Anaplasmosis, Anthrax, Appendicitis, Arcanobacteriumhaemolyticum infections, Argentine hemorrhagic fever, Ascariasis,Aspergillosis, Astrovirus infections, Athlete's foot, Babesiosis,Bacillus cereus infections, Bacterial meningitis, Bacterial pneumonia,Bacterial vaginosis (BV), Bacteroides infections, Balantidiasis,Baylisascaris infections, Bilharziosis, BK virus infections, Blackpiedra, Blastocystis hominis infections, Blastomycosis, Bolivianhemorrhagic fever, Borrelia infectionss (Borreliosis), Botulism (andInfant botulism), Bovine tapeworm, Brazilian hemorrhagic fever,Brucellosis, Burkholderia infections, Buruli ulcer, Calicivirusinfections (Norovirus and Sapovirus), Campylobacteriosis, Candidiasis(Candidosis), Canine tapeworm infections, Cat-scratch disease, ChagasDisease (American trypanosomiasis), Chancroid, Chickenpox, Chlamydiainfections, Chlamydia trachomatis infections, Chlamydophila pneumoniaeinfections, Cholera, Chromoblastomycosis, Climatic bubo, Clonorchiasis,Clostridium difficile infections, Coccidioidomycosis, Cold, Coloradotick fever (CTF), Common cold (Acute viral rhinopharyngitis; Acutecoryza), Condyloma acuminata, Conjunctivitis, Creutzfeldt-Jakob disease(CJD), Crimean-Congo hemorrhagic fever (CCHF), Cryptococcosis,Cryptosporidiosis, Cutaneous larva migrans (CLM), CutaneousLeishmaniosis, Cyclosporiasis, Cysticercosis, Cytomegalovirusinfections, Dengue fever, Dermatophytosis, Dientamoebiasis, Diphtheria,Diphyllobothriasis, Donavanosis, Dracunculiasis, Early summermeningoencephalitis (FSME), Ebola hemorrhagic fever, Echinococcosis,Ehrlichiosis, Enterobiasis (Pinworm infections), Enterococcusinfections, Enterovirus infections, Epidemic typhus, Epiglottitis,Epstein-Barr Virus Infectious Mononucleosis, Erythema infectiosum (Fifthdisease), Exanthem subitum, Fasciolopsiasis, Fasciolosis, Fatal familialinsomnia (FFI), Fifth disease, Filariasis, Fish poisoning (Ciguatera),Fish tapeworm, Flu, Food poisoning by Clostridium perfringens, Foxtapeworm, Free-living amebic infections, Fusobacterium infections, Gasgangrene, Geotrichosis, Gerstmann-Straussler-Scheinker syndrome (GSS),Giardiasis, Glanders, Gnathostomiasis, Gonorrhea, Granuloma inguinale(Donovanosis), Group A streptococcal infections, Group B streptococcalinfections, Haemophilus influenzae infections, Hand foot and mouthdisease (HFMD), Hantavirus Pulmonary Syndrome (HPS), Helicobacter pyloriinfections, Hemolytic-uremic syndrome (HUS), Hemorrhagic fever withrenal syndrome (HFRS), Henipavirus infections, Hepatitis A, Hepatitis B,Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, Herpes simplextype I, Herpes simplex type II, Herpes zoster, Histoplasmosis, Hollowwarts, Hookworm infections, Human bocavirus infections, Human ewingiiehrlichiosis, Human granulocytic anaplasmosis (HGA), Humanmetapneumovirus infections, Human monocytic ehrlichiosis, Humanpapillomavirus (HPV) infections, Human parainfluenza virus infections,Hymenolepiasis, Influenza, Isosporiasis, Japanese encephalitis, Kawasakidisease, Keratitis, Kingella kingae infections, Kuru, Lambliasis(Giardiasis), Lassa fever, Legionellosis (Legionnaires' disease, Pontiacfever), Leishmaniasis, Leprosy, Leptospirosis, Lice, Listeriosis, Lymeborreliosis, Lyme disease, Lymphatic filariasis (Elephantiasis),Lymphocytic choriomeningitis, Malaria, Marburg hemorrhagic fever (MHF),Marburg virus, Measles, Melioidosis (Whitmore's disease), Meningitis,Meningococcal disease, Metagonimiasis, Microsporidiosis, Miniaturetapeworm, Miscarriage (prostate inflammation), Molluscum contagiosum(MC), Mononucleosis, Mumps, Murine typhus (Endemic typhus), Mycetoma,Mycoplasma hominis, Mycoplasma pneumonia, Myiasis, Nappy/diaperdermatitis, Neonatal conjunctivitis (Ophthalmia neonatorum), Neonatalsepsis (Chorioamnionitis), Nocardiosis, Noma, Norwalk virus infections,Onchocerciasis (River blindness), Osteomyelitis, Otitis media,Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis,Paratyphus, Pasteurellosis, Pediculosis capitis (Head lice), Pediculosiscorporis (Body lice), Pediculosis pubis (Pubic lice, Crab lice), Pelvicinflammatory disease (PID), Pertussis (Whooping cough), Pfeiffer'sglandular fever, Plague, Pneumococcal infections, Pneumocystis pneumonia(PCP), Pneumonia, Polio (childhood lameness), Poliomyelitis, Porcinetapeworm, Prevotella infections, Primary amoebic meningoencephalitis(PAM), Progressive multifocal leukoencephalopathy, Pseudo-croup,Psittacosis, Q fever, Rabbit fever, Rabies, Rat-bite fever, Reiter'ssyndrome, Respiratory syncytial virus infections (RSV),Rhinosporidiosis, Rhinovirus infections, Rickettsial infections,Rickettsialpox, Rift Valley fever (RVF), Rocky mountain spotted fever(RMSF), Rotavirus infections, Rubella, Salmonella paratyphus, Salmonellatyphus, Salmonellosis, SARS (Severe Acute Respiratory Syndrome),Scabies, Scarlet fever, Schistosomiasis (Bilharziosis), Scrub typhus,Sepsis, Shigellosis (Bacillary dysentery), Shingles, Smallpox (Variola),Soft chancre, Sporotrichosis, Staphylococcal food poisoning,Staphylococcal infections, Strongyloidiasis, Syphilis, Taeniasis,Tetanus, Three-day fever, Tick-borne encephalitis, Tinea barbae(Barber's itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis(Ringworm of the Body), Tinea cruris (Jock itch), Tinea manuum (Ringwormof the Hand), Tinea nigra, Tinea pedis (Athlete's foot), Tinea unguium(Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis(Ocular Larva Migrans (OLM) and Visceral Larva Migrans (VLM)),Toxoplasmosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworminfections), Tripper, Trypanosomiasis (sleeping sickness), Tsutsugamushidisease, Tuberculosis, Tularemia, Typhus, Typhus fever, Ureaplasmaurealyticum infections, Vaginitis (Colpitis), Variant Creutzfeldt-Jakobdisease (vCJD, nvCJD), Venezuelan equine encephalitis, Venezuelanhemorrhagic fever, Viral pneumonia, Visceral Leishmaniosis, Warts, WestNile Fever, Western equine encephalitis, White piedra (Tinea blanca),Whooping cough, Yeast fungus spots, Yellow fever, Yersiniapseudotuberculosis infections, Yersiniosis, and Zygomycosis.

Cancer Diseases:

Preferably, diseases as mentioned herein are selected from cancer ortumour diseases which preferably include e.g. Acute lymphoblasticleukemia, Acute myeloid leukemia, Adrenocortical carcinoma, AIDS-relatedcancers, AIDS-related lymphoma, Anal cancer, Appendix cancer,Astrocytoma, Basal cell carcinoma, Bile duct cancer, Bladder cancer,Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma, Brainstemglioma, Brain tumor, cerebellar astrocytoma, cerebralastrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, visual pathway andhypothalamic glioma, Breast cancer, Bronchial adenomas/carcinoids,Burkitt lymphoma, childhood Carcinoid tumor, gastrointestinal Carcinoidtumor, Carcinoma of unknown primary, primary Central nervous systemlymphoma, childhood Cerebellar astrocytoma, childhood Cerebralastrocytoma/Malignant glioma, Cervical cancer, Childhood cancers,Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Chronicmyeloproliferative disorders, Colon Cancer, Cutaneous T-cell lymphoma,Desmoplastic small round cell tumor, Endometrial cancer, Ependymoma,Esophageal cancer, Ewing's sarcoma in the Ewing family of tumors,Childhood Extracranial germ cell tumor, Extragonadal Germ cell tumor,Extrahepatic bile duct cancer, Intraocular melanoma, Retinoblastoma,Gallbladder cancer, Gastric (Stomach) cancer, Gastrointestinal CarcinoidTumor, Gastrointestinal stromal tumor (GIST), extracranial,extragonadal, or ovarian Germ cell tumor, Gestational trophoblastictumor, Glioma of the brain stem, Childhood Cerebral Astrocytoma,Childhood Visual Pathway and Hypothalamic Glioma, Gastric carcinoid,Hairy cell leukemia, Head and neck cancer, Heart cancer, Hepatocellular(liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, childhoodHypothalamic and visual pathway glioma, Intraocular Melanoma, Islet CellCarcinoma (Endocrine Pancreas), Kaposi sarcoma, Kidney cancer (renalcell cancer), Laryngeal Cancer, Leukemias, acute lymphoblastic Leukemia,acute myeloid Leukemia, chronic lymphocytic Leukemia, chronicmyelogenous Leukemia, hairy cell Leukemia, Lip and Oral Cavity Cancer,Liposarcoma, Liver Cancer, Non-Small Cell Lung Cancer, Small Cell LungCancer, Lymphomas, AIDS-related Lymphoma, Burkitt Lymphoma, cutaneousT-Cell Lymphoma, Hodgkin Lymphoma, Non-Hodgkin Lymphomas, PrimaryCentral Nervous System Lymphoma, Waldenström Macroglobulinemia,Malignant Fibrous Histiocytoma of Bone/Osteosarcoma, ChildhoodMedulloblastoma, Melanoma, Intraocular (Eye) Melanoma, Merkel CellCarcinoma, Adult Malignant Mesothelioma, Childhood Mesothelioma,Metastatic Squamous Neck Cancer with Occult Primary, Mouth Cancer,Childhood Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma/PlasmaCell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Diseases, Chronic MyelogenousLeukemia, Adult Acute Myeloid Leukemia, Childhood Acute MyeloidLeukemia, Multiple Myeloma (Cancer of the Bone-Marrow), ChronicMyeloproliferative Disorders, Nasal cavity and paranasal sinus cancer,Nasopharyngeal carcinoma, Neuroblastoma, Oral Cancer, Oropharyngealcancer, Osteosarcoma/malignant fibrous histiocytoma of bone, Ovariancancer, Ovarian epithelial cancer (Surface epithelial-stromal tumor),Ovarian germ cell tumor, Ovarian low malignant potential tumor,Pancreatic cancer, islet cell Pancreatic cancer, Paranasal sinus andnasal cavity cancer, Parathyroid cancer, Penile cancer, Pharyngealcancer, Pheochromocytoma, Pineal astrocytoma, Pineal germinoma,childhood Pineoblastoma and supratentorial primitive neuroectodermaltumors, Pituitary adenoma, Plasma cell neoplasia/Multiple myeloma,Pleuropulmonary blastoma, Primary central nervous system lymphoma,Prostate cancer, Rectal cancer, Renal cell carcinoma (kidney cancer),Cancer of the Renal pelvis and ureter, Retinoblastoma, childhoodRhabdomyosarcoma, Salivary gland cancer, Sarcoma of the Ewing family oftumors, Kaposi Sarcoma, soft tissue Sarcoma, uterine Sarcoma, Sdzarysyndrome, Skin cancer (nonmelanoma), Skin cancer (melanoma), Merkel cellSkin carcinoma, Small intestine cancer, Squamous cell carcinoma,metastatic Squamous neck cancer with occult primary, childhoodSupratentorial primitive neuroectodermal tumor, Testicular cancer,Throat cancer, childhood Thymoma, Thymoma and Thymic carcinoma, Thyroidcancer, childhood Thyroid cancer, Transitional cell cancer of the renalpelvis and ureter, gestational Trophoblastic tumor, Urethral cancer,endometrial Uterine cancer, Uterine sarcoma, Vaginal cancer, childhoodVisual pathway and hypothalamic glioma, Vulvar cancer, Waldenstrimmacroglobulinemia, and childhood Wilms tumor (kidney cancer).

Allergies:

Preferably, diseases as mentioned herein are selected from allergieswhich preferably include e.g. pollen allergy (allergy against grasspollen, tree pollen (e.g. pollen of hazel, birch, alder, ash), flowerpollen, herb pollen (e.g. pollen of mugwort)), dust mite allergy, moldallergy (e.g. allergy against Acremonium, Aspergillus, Cladosporium,Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma, orAlternaria), pet allergy (allergy against animals; e.g against cats,dogs, horses), food allergy (e.g. allergy against fish (e.g. bass, cod,flounder), seafood (e.g. crab, lobster, shrimps), egg, wheat, nuts (e.g.peanuts, almonds, cashews, walnuts), soya, milk, etc.) or insect biteallergy (allergy against insect venom, e.g. venom of wasps, bees,hornets, ants, mosquitos, or ticks).

In a particularly preferred embodiment, an RNA comprising at least onemodification and comprising at least one open reading frame is used intreatment of prostate cancer.

Autoimmune Diseases:

According to another specific embodiment, diseases as defined hereincomprise autoimmune diseases as defined in the following. autoimmunediseases are preferably selected from Addison disease (autoimmuneadrenalitis, Morbus Addison), alopecia areata, Addison's anemia (MorbusBiermer), autoimmune hemolytic anemia (AIHA), autoimmune hemolyticanemia (AIHA) of the cold type (cold hemagglutinine disease, coldautoimmune hemolytic anemia (AIHA) (cold agglutinin disease), (CHAD)),autoimmune hemolytic anemia (AIHA) of the warm type (warm AIHA, warmautoimmune haemolytic anemia (AIHA)), autoimmune hemolyticDonath-Landsteiner anemia (paroxysmal cold hemoglobinuria),antiphospholipid syndrome (APS), atherosclerosis, autoimmune arthritis,arteriitis temporalis, Takayasu arteriitis (Takayasu's disease, aorticarch disease), temporal arteriitis/giant cell arteriitis, autoimmunechronic gastritis, autoimmune infertility, autoimmune inner ear disease(AIED), Basedow's disease (Morbus Basedow), Bechterew's disease (MorbusBechterew, ankylosing spondylitis, spondylitis ankylosans), Behcet'ssyndrome (Morbus Behcet), bowel disease including autoimmuneinflammatory bowel disease (including colitis ulcerosa (Morbus Crohn,Crohn's disease), cardiomyopathy, particularly autoimmunecardiomyopathy, idiopathic dilated cardiomyopathy (DCM), celiac spruedermatitis (gluten mediated enteropathia), chronic fatigue immunedysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy (CIDP), chronic polyarthritis, Churg-Strauss syndrome,cicatricial pemphigoid, Cogan syndrome, CREST syndrome (syndrom withCalcinosis cutis, Raynaud phenomenon, motility disorders of theesophagus, sklerodaktylia and teleangiectasia), Crohn's disease (MorbusCrohn, colitis ulcerosa), dermatitis herpetiformis during, dermatologicautoimmune diseases, dermatomyositis, Diabetes, Diabetes mellitus Type 1(type I diabetes, insuline dependent Diabetes mellitus), Diabetesmellitus Type 2 (type II diabetes), essential mixed cryoglobulinemia,essential mixed cryoglobulinemia, fibromyalgia, fibromyositis,Goodpasture syndrome (anti-GBM mediated glomerulonephritis), graftversus host disease, Guillain-Barr6 syndrome (GBM,Polyradikuloneuritis), haematologic autoimmune diseases, Hashimotothyroiditis, hemophilia, acquired hemophilia, hepatitis, autoimmunehepatitis, particularly autoimmune forms of chronic hepatitis,idiopathic pulmonary fibrosis (IPF), idiopathic thrombocytopenicpurpura, Immuno-thrombocytopenic purpura (Morbus Werlhof; ITP), IgAnephropathy, infertility, autoimmune infertility, juvenile rheumatoidarthritis (Morbus Still, Still syndrome), Lambert-Eaton syndrome, lichenplanus, lichen sclerosus, lupus erythematosus, systemic lupuserythematosus (SLE), lupus erythematosus (discoid form), Lyme arthritis(Lyme disease, borrelia arthritis), Méniere's disease (Morbus Ménierb);mixed connective tissue disease (MCTD), multiple sclerosis (MS,encephalomyelitis disseminate, Charcot's disease), Myasthenia gravis(myasthenia, MG), myosits, polymyositis, neural autoimmune diseases,neurodermitis, pemphigus vulgaris, bullous pemphigoid, scar formingpemphigoid; polyarteriitis nodosa (periarteiitis nodosa), polychondritis(panchondritis), polyglandular (autoimmune) syndrome (PGA syndrome,Schmidt's syndrome), Polymyalgia rheumatica, primary agammaglobulinemia,primary biliary cirrhosis PBC, primary autoimmune cholangitis),progressive systemic sclerosis (PSS), Psoriasis, Psoriasis vulgaris,Raynaud's phenomena, Reiter's syndrome (Morbus Reiter, urethralconjunctive synovial syndrome)), rheumatoid arthritis (RA, chronicpolyarthritis, rheumatic disease of the joints, rheumatic fever),sarcoidosis (Morbus Boeck, Besnier-Boeck-Schaumann disease), stiff-mansyndrome, Sclerodermia, Scleroderma, Sjögren's syndrome, sympatheticophtalmia; Transient gluten intolerance, transplanted organ rejection,uveitis, autoimmune uveiitis, Vasculitis, Vitiligo, (leucoderma, pieboldskin), and Wegner's disease (Morbus Wegner, Wegner's granulomatosis) Inthis context particularly preferred are inherited diseases selectedfrom: 1p36 deletion syndrome; 18p deletion syndrome; 21-hydroxylasedeficiency; 45,X (Turner syndrome); 47,XX,+21 (Down syndrome); 47,XXX(triple X syndrome); 47,XXY (Klinefelter syndrome); 47,XY,+21 (Downsyndrome); 47,XYY syndrome; 5-ALA dehydratase-deficient porphyria (ALAdehydratase deficiency); 5-aminolaevulinic dehydratase deficiencyporphyria (ALA dehydratase deficiency); 5p deletion syndrome (Cri duchat) 5p-syndrome (Cri du chat); A-T (ataxia-telangiectasia); AAT(alpha-1 antitrypsin deficiency); Absence of vas deferens (congenitalbilateral absence of vas deferens); Absent vasa (congenital bilateralabsence of vas deferens); aceruloplasminemia; ACG2 (achondrogenesis typeII); ACH (achondroplasia); Achondrogenesis type II; achondroplasia; Acidbeta-glucosidase deficiency (Gaucher disease type 1);Acrocephalosyndactyly (Apert) (Apert syndrome); acrocephalosyndactyly,type V (Pfeiffer syndrome); Acrocephaly (Apert syndrome); Acute cerebralGaucher's disease (Gaucher disease type 2); acute intermittentporphyria; ACY2 deficiency (Canavan disease); AD (Alzheimer's disease);Adelaide-type craniosynostosis (Muenke syndrome); Adenomatous PolyposisColi (familial adenomatous polyposis); Adenomatous Polyposis of theColon (familial adenomatous polyposis); ADP (ALA dehydratasedeficiency); adenylosuccinate lyase deficiency; Adrenal gland disorders(21-hydroxylase deficiency); Adrenogenital syndrome (21-hydroxylasedeficiency); Adrenoleukodystrophy; AIP (acute intermittent porphyria);AIS (androgen insensitivity syndrome); AKU (alkaptonuria); ALAdehydratase porphyria (ALA dehydratase deficiency); ALA-D porphyria (ALAdehydratase deficiency); ALA dehydratase deficiency; Alcaptonuria(alkaptonuria); Alexander disease; alkaptonuria; Alkaptonuric ochronosis(alkaptonuria); alpha-1 antitrypsin deficiency; alpha-1 proteinaseinhibitor (alpha-1 antitrypsin deficiency); alpha-1 related emphysema(alpha-1 antitrypsin deficiency); Alpha-galactosidase A deficiency(Fabry disease); ALS (amyotrophic lateral sclerosis); Alstrom syndrome;ALX (Alexander disease); Alzheimer disease; Amelogenesis Imperfecta;Amino levulinic acid dehydratase deficiency (ALA dehydratasedeficiency); Aminoacylase 2 deficiency (Canavan disease); amyotrophiclateral sclerosis; Anderson-Fabry disease (Fabry disease); androgeninsensitivity syndrome; Anemia; Anemia, hereditary sideroblastic(X-linked sideroblastic anemia); Anemia, sex-linked hypochromicsideroblastic (X-linked sideroblastic anemia); Anemia, splenic, familial(Gaucher disease); Angelman syndrome; Angiokeratoma Corporis Diffusum(Fabry's disease); Angiokeratoma diffuse (Fabry's disease); Angiomatosisretinae (von Hippel-Lindau disease); ANH1 (X-linked sideroblasticanemia); APC resistance, Leiden type (factor V Leiden thrombophilia);Apert syndrome; AR deficiency (androgen insensitivity syndrome); AR-CMT2ee (Charcot-Mare-Tooth disease, type 2); Arachnodactyly (Marfansyndrome); ARNSHL (Nonsyndromic deafness#autosomal recessive);Arthro-ophthalmopathy, hereditary progressive (Stickler syndrome#COL2A1); Arthrochalasis multiplex congenita (Ehlers-Danlossyndrome#arthrochalasia type); AS (Angelman syndrome); Asp deficiency(Canavan disease); Aspa deficiency (Canavan disease); Aspartoacylasedeficiency (Canavan disease); ataxia-telangiectasia;Autism-Dementia-Ataxia-Loss of Purposeful Hand Use syndrome (Rettsyndrome); autosomal dominant juvenile ALS (amyotrophic lateralsclerosis, type 4); Autosomal dominant opitz G/BBB syndrome (22q11.2deletion syndrome); autosomal recessive form of juvenile ALS type 3(Amyotrophic lateral sclerosis#type 2); Autosomal recessive nonsyndromichearing loss (Nonsyndromic deafness#autosomal recessive); AutosomalRecessive Sensorineural Hearing Impairment and Goiter (Pendredsyndrome); AxD (Alexander disease); Ayerza syndrome (primary pulmonaryhypertension); B variant of the Hexosaminidase GM2 gangliosidosis(Sandhoff disease); BANF (neurofibromatosis 2); Beare-Stevenson cutisgyrata syndrome; Benign paroxysmal peritonitis (Mediterranean fever,familial); Benjamin syndrome; beta thalassemia; BH4 Deficiency(tetrahydrobiopterin deficiency); Bilateral Acoustic Neurofibromatosis(neurofibromatosis 2); biotinidase deficiency; bladder cancer; Bleedingdisorders (factor V Leiden thrombophilia); Bloch-Sulzberger syndrome(incontinentia pigmenti); Bloom syndrome; Bone diseases; Bone marrowdiseases (X-linked sideroblastic anemia); Bonnevie-Ullrich syndrome(Turner syndrome); Bourneville disease (tuberous sclerosis); Bournevillephakomatosis (tuberous sclerosis); Brain diseases (prion disease);breast cancer; Birt-Hogg-Dub6 syndrome; Brittle bone disease(osteogenesis imperfecta); Broad Thumb-Hallux syndrome (Rubinstein-Taybisyndrome); Bronze Diabetes (hemochromatosis); Bronzed cirrhosis(hemochromatosis); Bulbospinal muscular atrophy, X-linked (Kennedydisease); Burger-Grutz syndrome (lipoprotein lipase deficiency,familial); CADASIL; CGD Chronic Granulomatous Disorder; Camptomelicdysplasia; Canavan disease; Cancer; Cancer Family syndrome (hereditarynonpolyposis colorectal cancer); Cancer of breast (breast cancer);Cancer of the bladder (bladder cancer); Carboxylase Deficiency,Multiple, Late-Onset (biotinidase deficiency); Cardiomyopathy (Noonansyndrome); Cat cry syndrome (Cri du chat); CAVD (congenital bilateralabsence of vas deferens); Caylor cardiofacial syndrome (22q11.2 deletionsyndrome); CBAVD (congenital bilateral absence of vas deferens); CeliacDisease; CEP (congenital erythropoietic porphyria); Ceramidetrihexosidase deficiency (Fabry disease); Cerebelloretinal Angiomatosis,familial (von Hippel-Lindau disease); Cerebral arteriopathy withsubcortical infarcts and leukoencephalopathy (CADASIL); Cerebralautosomal dominant ateriopathy with subcortical infarcts andleukoencephalopathy (CADASIL); Cerebral sclerosis (tuberous sclerosis);Cerebroatrophic Hyperammonemia (Rett syndrome); Cerebroside Lipidosissyndrome (Gaucher disease); CF (cystic fibrosis); CH (congenitalhypothyroidism); Charcot disease (amyotrophic lateral sclerosis);Charcot-Marie-Tooth disease; Chondrodystrophia (achondroplasia);Chondrodystrophy syndrome (achondroplasia); Chondrodystrophy withsensorineural deafness (otospondylomegaepiphyseal dysplasia);Chondrogenesis imperfecta (achondrogenesis, type II); Choreoathetosisself-mutilation hyperuricemia syndrome (Lesch-Nyhan syndrome); ClassicGalactosemia (galactosemia); Classical Ehlers-Danlos syndrome(Ehlers-Danlos syndrome#classical type); Classical Phenylketonuria(phenylketonuria); Cleft lip and palate (Stickler syndrome); Cloverleafskull with thanatophoric dwarfism (Thanatophoric dysplasia#type 2); CLS(Coffin-Lowry syndrome); CMT (Charcot-Marie-Tooth disease); Cockaynesyndrome; Coffin-Lowry syndrome; collagenopathy, types II and XI; ColonCancer, familial Nonpolyposis (hereditary nonpolyposis colorectalcancer); Colon cancer, familial (familial adenomatous polyposis);Colorectal Cancer; Complete HPRT deficiency (Lesch-Nyhan syndrome);Complete hypoxanthine-guanine phosphoribosy transferase deficiency(Lesch-Nyhan syndrome); Compression neuropathy (hereditary neuropathywith liability to pressure palsies); Congenital adrenal hyperplasia(21-hydroxylase deficiency); congenital bilateral absence of vasdeferens (Congenital absence of the vas deferens); Congenitalerythropoietic porphyria; Congenital heart disease; Congenitalhypomyelination (Charcot-Marie-Tooth disease#Type 1/Charcot-Marie-Toothdisease#Type 4); Congenital hypothyroidism; Congenital methemoglobinemia(Methemoglobinemia#Congenital methaemoglobinaemia); Congenitalosteosclerosis (achondroplasia); Congenital sideroblastic anaemia(X-linked sideroblastic anemia); Connective tissue disease; Conotruncalanomaly face syndrome (22q11.2 deletion syndrome); Cooley's Anemia (betathalassemia); Copper storage disease (Wilson disease); Copper transportdisease (Menkes disease); Coproporphyria, hereditary (hereditarycoproporphyria); Coproporphyrinogen oxidase deficiency (hereditarycoproporphyria); Cowden syndrome; CPO deficiency (hereditarycoproporphyria); CPRO deficiency (hereditary coproporphyria); CPXdeficiency (hereditary coproporphyria); Craniofacial dysarthrosis(Crouzon syndrome); Craniofacial Dysostosis (Crouzon syndrome);Cretinism (congenital hypothyroidism); Creutzfeldt-Jakob disease (priondisease); Cri du chat (Crohn's disease, fibrostenosing); Crouzonsyndrome; Crouzon syndrome with acanthosis nigricans(Crouzonodermoskeletal syndrome); Crouzonodermoskeletal syndrome; CS(Cockayne syndrome)(Cowden syndrome); Curschmann-Batten-Steinertsyndrome (myotonic dystrophy); cutis gyrata syndrome of Beare-Stevenson(Beare-Stevenson cutis gyrata syndrome); Disorder Mutation Chromosome;D-glycerate dehydrogenase deficiency (hyperoxaluria, primary); Dappledmetaphysis syndrome (spondyloepimetaphyseal dysplasia, Strudwick type);DAT—Dementia Alzheimer's type (Alzheimer disease); Genetichypercalciuria (Dent's disease); DBMD (muscular dystrophy, Duchenne andBecker types); Deafness with goiter (Pendred syndrome);Deafness-retinitis pigmentosa syndrome (Usher syndrome); Deficiencydisease, Phenylalanine Hydroxylase (phenylketonuria); Degenerative nervediseases; de Grouchy syndrome 1 (De Grouchy Syndrome); Dejerine-Sottassyndrome (Charcot-Marie-Tooth disease); Delta-aminolevulinatedehydratase deficiency porphyria (ALA dehydratase deficiency); Dementia(CADASIL); demyelinogenic leukodystrophy (Alexander disease);Dermatosparactic type of Ehlers-Danlos syndrome (Ehlers-Danlossyndrome#dermatosparaxis type); Dermatosparaxis (Ehlers-Danlossyndrome#dermatosparaxis type); developmental disabilities; dHMN(Amyotrophic lateral sclerosis#type 4); DHMN-V (distal spinal muscularatrophy, type V); DHTR deficiency (androgen insensitivity syndrome);Diffuse Globoid Body Sclerosis (Krabbe disease); DiGeorge syndrome;Dihydrotestosterone receptor deficiency (androgen insensitivitysyndrome); distal spinal muscular atrophy, type V; DM1 (Myotonicdystrophy#type1); DM2 (Myotonic dystrophy#type2); Down syndrome; DSMAV(distal spinal muscular atrophy, type V); DSN (Charcot-Marie-Toothdisease#type 4); DSS (Charcot-Marie-Tooth disease, type 4);Duchenne/Becker muscular dystrophy (muscular dystrophy, Duchenne andBecker types); Dwarf, achondroplastic (achondroplasia); Dwarf,thanatophoric (thanatophoric dysplasia); Dwarfism; Dwarfism-retinalatrophy-deafness syndrome (Cockayne syndrome); dysmyelinogenicleukodystrophy (Alexander disease); Dystrophia myotonica (myotonicdystrophy); dystrophia retinae pigmentosa-dysostosis syndrome (Ushersyndrome); Early-Onset familial alzheimer disease (EOFAD) (Alzheimerdisease); EDS (Ehlers-Danlos syndrome); Ehlers-Danlos syndrome;Ekman-Lobstein disease (osteogenesis imperfecta); Entrapment neuropathy(hereditary neuropathy with liability to pressure palsies); Epiloia(tuberous sclerosis); EPP (erythropoietic protoporphyria);Erythroblastic anemia (beta thalassemia); Erythrohepatic protoporphyria(erythropoietic protoporphyria); Erythroid 5-aminolevulinate synthetasedeficiency (X-linked sideroblastic anemia); Erythropoietic porphyria(congenital erythropoietic porphyria); Erythropoietic protoporphyria;Erythropoietic uroporphyria (congenital erythropoietic porphyria); Eyecancer (retinoblastoma FA—Friedreich ataxia); Fabry disease; Facialinjuries and disorders; Factor V Leiden thrombophilia; FALS (amyotrophiclateral sclerosis); familial acoustic neuroma (neurofibromatosis typeII); familial adenomatous polyposis; familial Alzheimer disease (FAD)(Alzheimer disease); familial amyotrophic lateral sclerosis (amyotrophiclateral sclerosis); familial dysautonomia; familial fat-inducedhypertriglyceridemia (lipoprotein lipase deficiency, familial); familialhemochromatosis (hemochromatosis); familial LPL deficiency (lipoproteinlipase deficiency, familial); familial nonpolyposis colon cancer(hereditary nonpolyposis colorectal cancer); familial paroxysmalpolyserositis (Mediterranean fever, familial); familial PCT (porphyriacutanea tarda); familial pressure sensitive neuropathy (hereditaryneuropathy with liability to pressure palsies); familial primarypulmonary hypertension (FPPH) (primary pulmonary hypertension); FamilialTurner syndrome (Noonan syndrome); familial vascular leukoencephalopathy(CADASIL); FAP (familial adenomatous polyposis); FD (familialdysautonomia); Female pseudo-Turner syndrome (Noonan syndrome);Ferrochelatase deficiency (erythropoietic protoporphyria); ferroportindisease (Haemochromatosis#type 4); Fever (Mediterranean fever,familial); FG syndrome; FGFR3-associated coronal synostosis (Muenkesyndrome); Fibrinoid degeneration of astrocytes (Alexander disease);Fibrocystic disease of the pancreas (cystic fibrosis); FMF(Mediterranean fever, familial); Folling disease (phenylketonuria);fra(X) syndrome (fragile X syndrome); fragile X syndrome; Fragilitasossium (osteogenesis imperfecta); FRAXA syndrome (fragile X syndrome);FRDA (Friedreich's ataxia); Friedreich ataxia (Friedreich's ataxia);Friedreich's ataxia; FXS (fragile X syndrome); G6PD deficiency;Galactokinase deficiency disease (galactosemia); Galactose-1-phosphateuridyl-transferase deficiency disease (galactosemia); galactosemia;Galactosylceramidase deficiency disease (Krabbe disease);Galactosylceramide lipidosis (Krabbe disease); galactosylcerebrosidasedeficiency (Krabbe disease); galactosylsphingosine lipidosis (Krabbedisease); GALC deficiency (Krabbe disease); GALT deficiency(galactosemia); Gaucher disease; Gaucher-like disease (pseudo-Gaucherdisease); GBA deficiency (Gaucher disease type 1); GD (Gaucher'sdisease); Genetic brain disorders; genetic emphysema (alpha-1antitrypsin deficiency); genetic hemochromatosis (hemochromatosis);Giant cell hepatitis, neonatal (Neonatal hemochromatosis); GLAdeficiency (Fabry disease); Glioblastoma, retinal (retinoblastoma);Glioma, retinal (retinoblastoma); globoid cell leukodystrophy (GCL, GLD)(Krabbe disease); globoid cell leukoencephalopathy (Krabbe disease);Glucocerebrosidase deficiency (Gaucher disease); Glucocerebrosidosis(Gaucher disease); Glucosyl cerebroside lipidosis (Gaucher disease);Glucosylceramidase deficiency (Gaucher disease); Glucosylceramidebeta-glucosidase deficiency (Gaucher disease); Glucosylceramidelipidosis (Gaucher disease); Glyceric aciduria (hyperoxaluria, primary);Glycine encephalopathy (Nonketotic hyperglycinemia); Glycolic aciduria(hyperoxaluria, primary); GM2 gangliosidosis, type 1 (Tay-Sachsdisease); Goiter-deafness syndrome (Pendred syndrome); Graefe-Ushersyndrome (Usher syndrome); Gronblad-Strandberg syndrome (pseudoxanthomaelasticum); Guenther porphyria (congenital erythropoietic porphyria);Gunther disease (congenital erythropoietic porphyria); Haemochromatosis(hemochromatosis); Hallgren syndrome (Usher syndrome); HarlequinIchthyosis; Hb S disease (sickle cell anemia); HCH (hypochondroplasia);HCP (hereditary coproporphyria); Head and brain malformations; Hearingdisorders and deafness; Hearing problems in children; HEF2A(hemochromatosis#type 2); HEF2B (hemochromatosis#type 2);Hematoporphyria (porphyria); Heme synthetase deficiency (erythropoieticprotoporphyria); Hemochromatoses (hemochromatosis); hemochromatosis;hemoglobin M disease (methemoglobinemia#beta-globin type); Hemoglobin Sdisease (sickle cell anemia); hemophilia; HEP (hepatoerythropoieticporphyria); hepatic AGT deficiency (hyperoxaluria, primary);hepatoerythropoietic porphyria; Hepatolenticular degeneration syndrome(Wilson disease); Hereditary arthro-ophthalmopathy (Stickler syndrome);Hereditary coproporphyria; Hereditary dystopic lipidosis (Fabrydisease); Hereditary hemochromatosis (HHC) (hemochromatosis); HereditaryInclusion Body Myopathy (skeletal muscle regeneration); Hereditaryiron-loading anemia (X-linked sideroblastic anemia); Hereditary motorand sensory neuropathy (Charcot-Marie-Tooth disease); Hereditary motorneuronopathy (spinal muscular atrophy); Hereditary motor neuronopathy,type V (distal spinal muscular atrophy, type V); Hereditary MultipleExostoses; Hereditary nonpolyposis colorectal cancer; Hereditaryperiodic fever syndrome (Mediterranean fever, familial); HereditaryPolyposis Coli (familial adenomatous polyposis); Hereditary pulmonaryemphysema (alpha-1 antitrypsin deficiency); Hereditary resistance toactivated protein C (factor V Leiden thrombophilia); Hereditary sensoryand autonomic neuropathy type III (familial dysautonomia); Hereditaryspastic paraplegia (infantile-onset ascending hereditary spasticparalysis); Hereditary spinal ataxia (Friedreich ataxia); Hereditaryspinal sclerosis (Friedreich ataxia); Herrick's anemia (sickle cellanemia); Heterozygous OSMED (Weissenbacher-Zweymiiller syndrome);Heterozygous otospondylomegaepiphyseal dysplasia(Weissenbacher-Zweymiller syndrome); HexA deficiency (Tay-Sachsdisease); Hexosaminidase A deficiency (Tay-Sachs disease);Hexosaminidase alpha-subunit deficiency (variant B) (Tay-Sachs disease);HFE-associated hemochromatosis (hemochromatosis); HGPS (Progeria);Hippel-Lindau disease (von Hippel-Lindau disease); HLAH(hemochromatosis); HMN V (distal spinal muscular atrophy, type V); HMSN(Charcot-Marie-Tooth disease); HNPCC (hereditary nonpolyposis colorectalcancer); HNPP (hereditary neuropathy with liability to pressurepalsies); homocystinuria; Homogentisic acid oxidase deficiency(alkaptonuria); Homogentisic acidura (alkaptonuria); Homozygousporphyria cutanea tarda (hepatoerythropoietic porphyria); HP1(hyperoxaluria, primary); HP2 (hyperoxaluria, primary); HPA(hyperphenylalaninemia); HPRT—Hypoxanthine-guaninephosphoribosyltransferase deficiency (Lesch-Nyhan syndrome); HSAN typeIII (familial dysautonomia); HSAN3 (familial dysautonomia); HSN-III(familial dysautonomia); Human dermatosparaxis (Ehlers-Danlossyndrome#dermatosparaxis type); Huntington's disease; Hutchinson-Gilfordprogeria syndrome (progeria); Hyperandrogenism, nonclassic type, due to21-hydroxylase deficiency (21-hydroxylase deficiency);Hyperchylomicronemia, familial (lipoprotein lipase deficiency,familial); hyperglycinemia with ketoacidosis and leukopenia (propionicacidemia); Hyperlipoproteinemia type I (lipoprotein lipase deficiency,familial); hyperoxaluria, primary; hyperphenylalaninaemia(hyperphenylalaninemia); hyperphenylalaninemia; Hypochondrodysplasia(hypochondroplasia); hypochondrogenesis; hypochondroplasia; Hypochromicanemia (X-linked sideroblastic anemia); Hypocupremia, congenital; Menkessyndrome); hypoxanthine phosphoribosyltransferse (HPRT) deficiency(Lesch-Nyhan syndrome); IAHSP (infantile-onset ascending hereditaryspastic paralysis); idiopathic hemochromatosis (hemochromatosis, type3); Idiopathic neonatal hemochromatosis (hemochromatosis, neonatal);Idiopathic pulmonary hypertension (primary pulmonary hypertension);Immune system disorders (X-linked severe combined immunodeficiency);Incontinentia Pigmenti; Infantile cerebral Gaucher's disease (Gaucherdisease type 2); Infantile Gaucher disease (Gaucher disease type 2);infantile-onset ascending hereditary spastic paralysis; Infertility;inherited emphysema (alpha-1 antitrypsin deficiency); Inherited humantransmissible spongiform encephalopathies (prion disease); inheritedtendency to pressure palsies (hereditary neuropathy with liability topressure palsies); Insley-Astley syndrome (otospondylomegaepiphysealdysplasia); Intermittent acute porphyria syndrome (acute intermittentporphyria); Intestinal polyposis-cutaneous pigmentation syndrome(Peutz-Jeghers syndrome); IP (incontinentia pigmenti); Iron storagedisorder (hemochromatosis); Isodicentric 15 (idicl5); Isolated deafness(nonsyndromic deafness); Jackson-Weiss syndrome; JH(Haemochromatosis#type 2); Joubert syndrome; JPLS (Juvenile PrimaryLateral Sclerosis); juvenile amyotrophic lateral sclerosis (Amyotrophiclateral sclerosis#type 2); Juvenile gout, choreoathetosis, mentalretardation syndrome (Lesch-Nyhan syndrome); juvenile hyperuricemiasyndrome (Lesch-Nyhan syndrome); JWS (Jackson-Weiss syndrome); KD(X-linked spinal-bulbar muscle atrophy); Kennedy disease (X-linkedspinal-bulbar muscle atrophy); Kennedy spinal and bulbar muscularatrophy (X-linked spinal-bulbar muscle atrophy); Kerasin histiocytosis(Gaucher disease); Kerasin lipoidosis (Gaucher disease); Kerasinthesaurismosis (Gaucher disease); ketotic glycinemia (propionicacidemia); ketotic hyperglycinemia (propionic acidemia); Kidney diseases(hyperoxaluria, primary); Klinefelter syndrome; Klinefelter's syndrome;Kniest dysplasia; Krabbe disease; Lacunar dementia (CADASIL);Langer-Saldino achondrogenesis (achondrogenesis, type II);Langer-Saldino dysplasia (achondrogenesis, type II); Late-onsetAlzheimer disease (Alzheimer disease#type 2); Late-onset familialAlzheimer disease (AD2) (Alzheimer disease#type 2); late-onset Krabbedisease (LOKD) (Krabbe disease); Learning Disorders (Learningdisability); Lentiginosis, perioral (Peutz-Jeghers syndrome);Lesch-Nyhan syndrome; Leukodystrophies; leukodystrophy with Rosenthalfibers (Alexander disease); Leukodystrophy, spongiform (Canavandisease); LFS (Li-Fraumeni syndrome); Li-Fraumeni syndrome; Lipase Ddeficiency (lipoprotein lipase deficiency, familial); LIPD deficiency(lipoprotein lipase deficiency, familial); Lipidosis, cerebroside(Gaucher disease); Lipidosis, ganglioside, infantile (Tay-Sachsdisease); Lipoid histiocytosis (kerasin type) (Gaucher disease);lipoprotein lipase deficiency, familial; Liver diseases (galactosemia);Lou Gehrig disease (amyotrophic lateral sclerosis); Louis-Bar syndrome(ataxia-telangiectasia); Lynch syndrome (hereditary nonpolyposiscolorectal cancer); Lysyl-hydroxylase deficiency (Ehlers-Danlossyndrome#kyphoscoliosis type); Machado-Joseph disease (Spinocerebellarataxia#type 3); Male breast cancer (breast cancer); Male genitaldisorders; Male Turner syndrome (Noonan syndrome); Malignant neoplasm ofbreast (breast cancer); malignant tumor of breast (breast cancer);Malignant tumor of urinary bladder (bladder cancer); Mammary cancer(breast cancer); Marfan syndrome 15; Marker X syndrome (fragile Xsyndrome); Martin-Bell syndrome (fragile X syndrome); McCune-Albrightsyndrome; McLeod syndrome; MEDNIK; Mediterranean Anemia (betathalassemia); Mediterranean fever, familial; Mega-epiphyseal dwarfism(otospondylomegaepiphyseal dysplasia); Menkea syndrome (Menkessyndrome); Menkes syndrome; Mental retardation with osteocartilaginousabnormalities (Coffin-Lowry syndrome); Metabolic disorders; Metatropicdwarfism, type II (Kniest dysplasia); Metatropic dysplasia type II(Kniest dysplasia); Methemoglobinemia#beta-globin type; methylmalonicacidemia; MFS (Marfan syndrome); MHAM (Cowden syndrome); MK (Menkessyndrome); Micro syndrome; Microcephaly; MMA (methylmalonic acidemia);MNK (Menkes syndrome); Monosomy 1p36 syndrome (1p36 deletion syndrome);monosomy X (Turner syndrome); Motor neuron disease, amyotrophic lateralsclerosis (amyotrophic lateral sclerosis); Movement disorders;Mowat-Wilson syndrome; Mucopolysaccharidosis (MPS I); Mucoviscidosis(cystic fibrosis); Muenke syndrome; Multi-Infarct dementia (CADASIL);Multiple carboxylase deficiency, late-onset (biotinidase deficiency);Multiple hamartoma syndrome (Cowden syndrome); Multipleneurofibromatosis (neurofibromatosis); Muscular dystrophy; Musculardystrophy, Duchenne and Becker type; Myotonia atrophica (myotonicdystrophy); Myotonia dystrophica (myotonic dystrophy); myotonicdystrophy; Myxedema, congenital (congenital hypothyroidism);Nance-Insley syndrome (otospondylomegaepiphyseal dysplasia);Nance-Sweeney chondrodysplasia (otospondylomegaepiphyseal dysplasia);NBIA1 (pantothenate kinase-associated neurodegeneration); Neill-Dingwallsyndrome (Cockayne syndrome); Neuroblastoma, retinal (retinoblastoma);Neurodegeneration with brain iron accumulation type 1 (pantothenatekinase-associated neurodegeneration); Neurofibromatosis type I;Neurofibromatosis type II; Neurologic diseases; Neuromuscular disorders;neuronopathy, distal hereditary motor, type V (Distal spinal muscularatrophy#type V); neuronopathy, distal hereditary motor, with pyramidalfeatures (Amyotrophic lateral sclerosis#type 4); NF (neurofibromatosis);Niemann-Pick (Niemann-Pick disease); Noack syndrome (Pfeiffer syndrome);Nonketotic hyperglycinemia (Glycine encephalopathy); Non-neuronopathicGaucher disease (Gaucher disease type 1); Non-phenylketonurichyperphenylalaninemia (tetrahydrobiopterin deficiency); nonsyndromicdeafness; Noonan syndrome; Norrbottnian Gaucher disease (Gaucher diseasetype 3); Ochronosis (alkaptonuria); Ochronotic arthritis (alkaptonuria);OI (osteogenesis imperfecta); OSMED (otospondylomegaepiphysealdysplasia); osteogenesis imperfecta; Osteopsathyrosis (osteogenesisimperfecta); Osteosclerosis congenita (achondroplasia);Oto-spondylo-megaepiphyseal dysplasia (otospondylomegaepiphysealdysplasia); otospondylomegaepiphyseal dysplasia; Oxalosis(hyperoxaluria, primary); Oxaluria, primary (hyperoxaluria, primary);pantothenate kinase-associated neurodegeneration; Patau Syndrome(Trisomy 13); PBGD deficiency (acute intermittent porphyria); PCCdeficiency (propionic acidemia); PCT (porphyria cutanea tarda); PDM(Myotonic dystrophy#type 2); Pendred syndrome; Periodic disease(Mediterranean fever, familial); Periodic peritonitis (Mediterraneanfever, familial); Periorificial lentiginosis syndrome (Peutz-Jegherssyndrome); Peripheral nerve disorders (familial dysautonomia);Peripheral neurofibromatosis (neurofibromatosis 1); Peroneal muscularatrophy (Charcot-Marie-Tooth disease); peroxisomal alanine:glyoxylateaminotransferase deficiency (hyperoxaluria, primary); Peutz-Jegherssyndrome; Pfeiffer syndrome; Phenylalanine hydroxylase deficiencydisease (phenylketonuria); phenylketonuria; Pheochromocytoma (vonHippel-Lindau disease); Pierre Robin syndrome with fetalchondrodysplasia (Weissenbacher-Zweymiiller syndrome); Pigmentarycirrhosis (hemochromatosis); PJS (Peutz-Jeghers syndrome); PKAN(pantothenate kinase-associated neurodegeneration); PKU(phenylketonuria); Plumboporphyria (ALA deficiency porphyria); PMA(Charcot-Marie-tooth disease); polyostotic fibrous dysplasia(McCune-Albright syndrome); polyposis coli (familial adenomatouspolyposis); polyposis, hamartomatous intestinal (Peutz-Jegherssyndrome); polyposis, intestinal, II (Peutz-Jeghers syndrome);polyps-and-spots syndrome (Peutz-Jeghers syndrome); Porphobilinogensynthase deficiency (ALA deficiency porphyria); porphyria; porphyrindisorder (porphyria); PPH (primary pulmonary hypertension); PPOXdeficiency (variegate porphyria); Prader-Labhart-Willi syndrome(Prader-Willi syndrome); Prader-Willi syndrome; presenile and seniledementia (Alzheimer disease); primary hemochromatosis (hemochromatosis);primary hyperuricemia syndrome (Lesch-Nyhan syndrome); primary pulmonaryhypertension; primary senile degenerative dementia (Alzheimer disease);prion disease; procollagen type EDS VII, mutant (Ehlers-Danlossyndrome#arthrochalasia type); progeria (Hutchinson Gilford ProgeriaSyndrome); Progeria-like syndrome (Cockayne syndrome); progeroid nanism(Cockayne syndrome); progressive chorea, chronic hereditary (Huntington)(Huntington's disease); progressive muscular atrophy (spinal muscularatrophy); progressively deforming osteogenesis imperfecta with normalsclerae (Osteogenesis imperfecta#type III); PROMM (Myotonicdystrophy#type 2); propionic academia; propionyl-CoA carboxylasedeficiency (propionic acidemia); protein C deficiency; protein Sdeficiency; protoporphyria (erythropoietic protoporphyria);protoporphyrinogen oxidase deficiency (variegate porphyria); proximalmyotonic dystrophy (Myotonic dystrophy#type 2); proximal myotonicmyopathy (Myotonic dystrophy#type 2); pseudo-Gaucher disease;pseudo-Ullrich-Turner syndrome (Noonan syndrome); pseudoxanthomaelasticum; psychosine lipidosis (Krabbe disease); pulmonary arterialhypertension (primary pulmonary hypertension); pulmonary hypertension(primary pulmonary hypertension); PWS (Prader-Willi syndrome);PXE—pseudoxanthoma elasticum (pseudoxanthoma elasticum); Rb(retinoblastoma); Recklinghausen disease, nerve (neurofibromatosis 1);Recurrent polyserositis (Mediterranean fever, familial); Retinaldisorders; Retinitis pigmentosa-deafness syndrome (Usher syndrome);Retinoblastoma; Rett syndrome; RFALS type 3 (Amyotrophic lateralsclerosis#type 2); Ricker syndrome (Myotonic dystrophy#type 2);Riley-Day syndrome (familial dysautonomia); Roussy-Levy syndrome(Charcot-Marie-Tooth disease); RSTS (Rubinstein-Taybi syndrome); RTS(Rett syndrome) (Rubinstein-Taybi syndrome); RTT (Rett syndrome);Rubinstein-Taybi syndrome; Sack-Barabas syndrome (Ehlers-Danlossyndrome, vascular type); SADDAN; sarcoma family syndrome of Li andFraumeni (Li-Fraumeni syndrome); sarcoma, breast, leukemia, and adrenalgland (SBLA) syndrome (Li-Fraumeni syndrome); SBLA syndrome (Li-Fraumenisyndrome); SBMA (X-linked spinal-bulbar muscle atrophy); SCD (sicklecell anemia); Schwannoma, acoustic, bilateral (neurofibromatosis 2);SCIDX1 (X-linked severe combined immunodeficiency); sclerosis tuberosa(tuberous sclerosis); SDAT (Alzheimer disease); SED congenita(spondyloepiphyseal dysplasia congenita); SED Strudwick(spondyloepimetaphyseal dysplasia, Strudwick type); SEDc(spondyloepiphyseal dysplasia congenita); SEMD, Strudwick type(spondyloepimetaphyseal dysplasia, Strudwick type); senile dementia(Alzheimer disease#type 2); severe achondroplasia with developmentaldelay and acanthosis nigricans (SADDAN); Shprintzen syndrome (22q11.2deletion syndrome); sickle cell anemia; skeleton-skin-brain syndrome(SADDAN); Skin pigmentation disorders; SMA (spinal muscular atrophy);SMED, Strudwick type (spondyloepimetaphyseal dysplasia, Strudwick type);SMED, type I (spondyloepimetaphyseal dysplasia, Strudwick type); SmithLemli Opitz Syndrome; South-African genetic porphyria (variegateporphyria); spastic paralysis, infantile onset ascending(infantile-onset ascending hereditary spastic paralysis); Speech andcommunication disorders; sphingolipidosis, Tay-Sachs (Tay-Sachsdisease); spinal-bulbar muscular atrophy; spinal muscular atrophy;spinal muscular atrophy, distal type V (Distal spinal muscularatrophy#type V); spinal muscular atrophy, distal, with upper limbpredominance (Distal spinal muscular atrophy#type V); spinocerebellarataxia; spondyloepimetaphyseal dysplasia, Strudwick type;spondyloepiphyseal dysplasia congenital; spondyloepiphyseal dysplasia(collagenopathy, types II and XI); spondylometaepiphyseal dysplasiacongenita, Strudwick type (spondyloepimetaphyseal dysplasia, Strudwicktype); spondylometaphyseal dysplasia (SMD) (spondyloepimetaphysealdysplasia, Strudwick type); spondylometaphyseal dysplasia, Strudwicktype (spondyloepimetaphyseal dysplasia, Strudwick type); spongydegeneration of central nervous system (Canavan disease); spongydegeneration of the brain (Canavan disease); spongy degeneration ofwhite matter in infancy (Canavan disease); sporadic primary pulmonaryhypertension (primary pulmonary hypertension); SSB syndrome (SADDAN);steely hair syndrome (Menkes syndrome); Steinert disease (myotonicdystrophy); Steinert myotonic dystrophy syndrome (myotonic dystrophy);Stickler syndrome; stroke (CADASIL); Strudwick syndrome(spondyloepimetaphyseal dysplasia, Strudwick type); subacuteneuronopathic Gaucher disease (Gaucher disease type 3); Swedish geneticporphyria (acute intermittent porphyria); Swedish porphyria (acuteintermittent porphyria); Swiss cheese cartilage dysplasia (Kniestdysplasia); Tay-Sachs disease; TD—thanatophoric dwarfism (thanatophoricdysplasia); TD with straight femurs and cloverleaf skull (thanatophoricdysplasia#Type 2); Telangiectasia, cerebello-oculocutaneous(ataxia-telangiectasia); Testicular feminization syndrome (androgeninsensitivity syndrome); tetrahydrobiopterin deficiency; TFM—testicularfeminization syndrome (androgen insensitivity syndrome); thalassemiaintermedia (beta thalassemia); Thalassemia Major (beta thalassemia);thanatophoric dysplasia; thiamine-responsive megaloblastic anemia withdiabetes mellitus and sensorineural deafness; Thrombophilia due todeficiency of cofactor for activated protein C, Leiden type (factor VLeiden thrombophilia); Thyroid disease; Tomaculous neuropathy(hereditary neuropathy with liability to pressure palsies); Total HPRTdeficiency (Lesch-Nyhan syndrome); Total hypoxanthine-guaninephosphoribosyl transferase deficiency (Lesch-Nyhan syndrome); Tourette'sSyndrome; Transmissible dementias (prion disease); Transmissiblespongiform encephalopathies (prion disease); Treacher Collins syndrome;Trias fragilitis ossium (osteogenesis imperfecta#Type I); triple Xsyndrome; Triplo X syndrome (triple X syndrome); Trisomy 21 (Downsyndrome); Trisomy X (triple X syndrome); Troisier-Hanot-Chauffardsyndrome (hemochromatosis); TS (Turner syndrome); TSD (Tay-Sachsdisease); TSEs (prion disease); tuberose sclerosis (tuberous sclerosis);tuberous sclerosis; Turner syndrome; Turner syndrome in female with Xchromosome (Noonan syndrome); Turner's phenotype, karyotype normal(Noonan syndrome); Turner's syndrome (Turner syndrome); Turner-likesyndrome (Noonan syndrome); Type 2 Gaucher disease (Gaucher disease type2); Type 3 Gaucher disease (Gaucher disease type 3);UDP-galactose-4-epimerase deficiency disease (galactosemia); UDP glucose4-epimerase deficiency disease (galactosemia); UDP glucosehexose-1-phosphate uridylyltransferase deficiency (galactosemia);Ullrich-Noonan syndrome (Noonan syndrome); Ullrich-Turner syndrome(Turner syndrome); Undifferentiated deafness (nonsyndromic deafness);UPS deficiency (acute intermittent porphyria); Urinary bladder cancer(bladder cancer); UROD deficiency (porphyria cutanea tarda);Uroporphyrinogen decarboxylase deficiency (porphyria cutanea tarda);Uroporphyrinogen synthase deficiency (acute intermittent porphyria);UROS deficiency (congenital erythropoietic porphyria); Usher syndrome;UTP hexose-1-phosphate uridylyltransferase deficiency (galactosemia);Van Bogaert-Bertrand syndrome (Canavan disease); Van der Hoeve syndrome(osteogenesis imperfecta#Type I); variegate porphyria; Velocardiofacialsyndrome (22q11.2 deletion syndrome); VHL syndrome (von Hippel-Lindaudisease); Vision impairment and blindness (Alstrom syndrome); VonBogaert-Bertrand disease (Canavan disease); von Hippel-Lindau disease;Von Recklenhausen-Applebaum disease (hemochromatosis); vonRecklinghausen disease (neurofibromatosis 1); VP (variegate porphyria);Vrolik disease (osteogenesis imperfecta); Waardenburg syndrome; WarburgSjo Fledelius Syndrome (Micro syndrome); WD (Wilson disease);Weissenbacher-Zweymiiller syndrome; Wilson disease; Wilson's disease(Wilson disease); Wolf-Hirschhorn syndrome; Wolff Periodic disease(Mediterranean fever, familial); WZS (Weissenbacher-Zweymiillersyndrome); Xeroderma Pigmentosum; X-linked mental retardation andmacroorchidism (fragile X syndrome); X-linked primary hyperuricemia(Lesch-Nyhan syndrome); X-linked severe combined immunodeficiency;X-linked sideroblastic anemia; X-linked spinal-bulbar muscle atrophy(Kennedy disease); X-linked uric aciduria enzyme defect (Lesch-Nyhansyndrome); X-SCID (X-linked severe combined immunodeficiency); XLSA(X-linked sideroblastic anemia); XSCID (X-linked severe combinedimmunodeficiency); XXX syndrome (triple X syndrome); XXXX syndrome (48,XXXX); XXXXX syndrome (49, XXXXX); XXY syndrome (Klinefelter syndrome);XXY trisomy (Klinefelter syndrome); XYY karyotype (47,XYY syndrome); XYYsyndrome (47,XYY syndrome); and YY syndrome (47,XYY syndrome).

In a further preferred aspect, the modified RNA as defined herein or thecomposition comprising a plurality of modified RNA's as defined hereinmay be used for the preparation of a pharmaceutical composition,particularly for purposes as defined herein, preferably for the use ingene therapy or genetic vaccination in the treatment of diseases asdefined herein.

The pharmaceutical composition may furthermore be used in gene therapyor genetic vaccination particularly in the treatment of a disease or adisorder, preferably as defined herein.

According to a further aspect, the present invention also provides kits,particularly kits of parts. Such kits, particularly kits of parts,typically comprise as components alone or in combination with furthercomponents as defined herein at least one modified RNA as definedherein, the pharmaceutical composition or vaccine comprising themodified RNA. The at least one modified RNA as defined herein, isoptionally in combination with further components as defined herein,whereby the at least one modified RNA according to the invention isprovided separately (first part of the kit) from at least one other partof the kit comprising one or more other components. The pharmaceuticalcomposition may e.g. occur in one or different parts of the kit. As anexample, e.g. at least one part of the kit may comprise at least onemodified RNA as defined herein, and at least one further part of the kitat least one other component as defined herein, e.g. at least one otherpart of the kit may comprise at least one pharmaceutical composition ora part thereof, e.g. at least one part of the kit may comprise themodified RNA as defined herein, at least one further part of the kit atleast one other component as defined herein, at least one further partof the kit at least one component of the pharmaceutical composition orthe pharmaceutical composition as a whole, and at least one further partof the kit e.g. at least one pharmaceutical carrier or vehicle, etc. Incase the kit or kit of parts comprises a plurality of modified RNAmolecules, one component of the kit can comprise only one, several orall modifiedRNA molecules comprised in the kit. In an alternativeembodiment every/each modified RNA may be comprised in adifferent/separate component of the kit such that each component forms apart of the kit. Also, more than one modified RNA may be comprised in afirst component as part of the kit, whereas one or more other (second,third etc.) components (providing one or more other parts of the kit)may either contain one or more than one modified RNA's, which may beidentical or partially identical or different from the first component.The kit or kit of parts may furthermore contain technical instructionswith information on the administration and dosage of the modified RNA,the pharmaceutical composition or of any of its components or parts,e.g. if the kit is prepared as a kit of parts.

Preferably, the kit according to the invention comprises the modifiedRNA, preferably in lyophilized form and a suitable vector forreconstitution of the modified RNA. In a preferred embodiment themodified RNA is provided in a container, preferably in a container, inwhich the modified RNA is resolubilized. Preferably, the container canbe connected to a needle-free injection device, e.g. for filling adisposable syringe of a the needle-free injection device. Additionally,the invention relates to a method for enhancing the (localized)expression of RNA-encoded peptides or proteins in the dermis or muscle(of a mammal) comprising administering the modified RNA as definedherein by jet injection.

Furthermore, the present invention provides a method for treating orpreventing a disease or disorder comprising administration of a modifiedRNA as defined herein by jet injection to a subject in need thereof,particularly a human patient.

The method of treating or preventing a disorder according to the presentinvention is preferably a vaccination method and/or a gene therapymethod as described above.

Examples

The examples shown in the following are merely illustrative and shalldescribe the present invention in a further way. These examples shallnot be construed to limit the present invention thereto.

1. Expression of Luciferase In Vivo Preparation of DNA-Templates

A vector for in vitro transcription was constructed containing a T7promoter followed by a GC-enriched sequence coding for Photinus pyralisluciferase (PpLuc(GC)), a mutated 3′-UTR of alpha globin (muag), an A64poly(A) sequence, a poly(C) sequence (C30) and a histone stem-loopsequence (histone-SL):

SEQ ID No. 46 (FIG. 4): PpLuc(GC)-muag-A64-C30-histoneSL (R1265)

For comparison a vector was constructed containing a T7 promoterfollowed by a wild type sequence coding for Photinus pyralis luciferase(PpLuc(wt)) and a A30 poly(A) sequence:

SEQ ID No. 47 (FIG. 5): PpLuc(wt)-A30 (R2652)

To determine the effect of the used modifications following vectors wereconstructed:

SEQ ID No. 48 (FIG. 6): PpLuc(wt)-A64 (R3454) SEQ ID No. 49 (FIG. 7):PpLuc(GC)-A64-C30-HistoneSL (R2462)

SEQ ID No. 50 (FIG. 8): PpLuc(GC)-muag-A64-C30 (R1256)SEQ ID No. 51 (FIG. 9): PpLuc(nat)-muag-A64-C30-HistoneSL (R2393)

For Further Experiments, the Following Vector Encoding the G Protein ofRabies Virus (RAV-G) was Constructed:

Simarly to the vector PpLuc(GC)-muag-A64-C30-histoneSL (R1265) encodingluciferase, RAV-G(GC)-muag-A64-C30-histoneSL (R2403) contains a T7promoter followed by a GC-enriched sequence encoding the G protein ofRabies virus, a mutated 3′-UTR of alpha globin (muag), an A64 poly(A)sequence, a poly(C) sequence (C30) and a histone stem-loop sequence(histone-SL):

SEQ ID No. 52 (FIG. 10): RAV-G(GC)-muag-A64-C30-histoneSL (R2403)For Further Experiments, the Following Vectors Encoding Murine EPO wereConstructed:

A vector for in vitro transcription was constructed containing a T7promoter followed by the 5′-TOP-UTR sequence of HSD17B4, a GC-enrichedsequence encoding EPO (EPO(GC)), the 3′-UTR sequence of albumin(albumin7), an A64 poly(A) sequence, a poly(C) sequence (C30) and ahistone stem-loop sequence (histone-SL):

SEQ ID No. 54 (FIG. 12): HSD17B4-EPO(GC)-albumin7-A64-C30-histoneSL(R3135)

For comparison, a vector was constructed containing a T7 promoterfollowed by a wild type sequence encoding EPO (EPO(wt)) and an A30poly(A) sequence:

SEQ ID No. 53 (FIG. 11): EPO(wt)-A30 (R3513) In Vitro Transcription

The DNA-templates according to Example 1 were linearized and transcribedin vitro using T7-Polymerase. The DNA-template was then digested byDNase-treatment. mRNA transcripts contained a 5′-CAP structure (mCap)obtained by adding an excess ofN7-Methyl-Guanosine-5′-Triphosphate-5′-Guanosine to the transcriptionreaction. mRNA thus obtained was purified and resuspended in water.

Expression of Luciferase In Vivo:

For determining luciferase expression in vivo, guinea pigs wereintradermally injected with the indicated amounts of naked mRNA by jetinjection.

After 24 h, the animals were sacrificed and the samples (ear, skin fromthe back or muscle) were collected, frozen at −78° C. and lysed for 3minutes at full speed in a tissue lyser (Qiagen, Hilden, Germany).Afterwards 600 l of lysis buffer (25 mM Tris, pH 7.5 (HCl), 2 mM EDTA,10% glycerol, 1% Triton X-100, 2 mM DTT, 1 mM PMSF) were added and theresulting solutions were subjected another 6 minutes at full speed inthe tissue lyser. After 10 minutes of centrifugation at 13,500 rpm at 4°C., the supernatants were mixed with luciferin buffer (25 mMGlycylglycin, 15 mM MgSO4, 5 mM ATP, 62.5 μM luciferin) and luminiscencewas detected using a luminometer (Lumat LB 9507 (Berthold Technologies,Bad Wildbad, Germany)).

Results:

As a first result, it has been found that expression of luciferaseencoded by a modified mRNA is strongly enhanced in guinea pigs by jetinjection compared to conventional intradermal needle injection (seeFIG. 1).

Moreover, it has surprisingly been found that the saturation level ofexpression could be increased by jet injection compared toconventionally injected mRNA by needle-syringe (see FIG. 2).

Unexpectedly, it could further be shown that the expression ofluciferase from a modified mRNA (PpLuc(GC)) is significantly moreenhanced by jet injection than the expression of luciferase from anunmodified reference mRNA (PpLuc(wt); see FIG. 3).

TABLE 1 Summary of the measured increase in protein expression Effect ofjet Luciferase Effect of jet Effect of injection and expressioninjection modification modification PpLuc(GC) 5 275476 123 μg - conv.PpLuc(GC) 5 2267492 1013 μg - Jet PpLuc(wt) 5 2238 μg - conv. PpLuc(wt)5 4469 2 μg - Jet PpLuc(GC) 80 1926120 117 μg - conv. PpLuc(GC) 8017932040 1089 μg - Jet PpLuc(wt) 80 16471 μg - conv. PpLuc(wt) 80 270861.6 μg - Jet mock 1107

Thus, the modification of RNA and the administration of said modifiedRNA by jet injection act synergistically in increasing the expression ofthe protein encoded by the RNA (see Table 1).

In further experiments, it has been found that the additional increaseof expression, which was achieved by jet injection using the modifiedRNA according to the invention (see Table 2 below; e.g. R1265, R2462,R1256, R2393), as compared to using an unmodified reference RNA (seeTable 2; R2652), was achieved by using distinct modified RNAs, eachcomprising one or more of several distinct modifications. Table 2 showsthe fold increase in protein expression achieved by administration of agiven RNA (e.g. R1265) normalized to the protein expression achieved byadministration of the respective RNA by conventional intradermalinjection using a needle. For instance, the expression of modified RNAR1265 is increased by jet injection by a factor of 8.23 (as compared tothe expression obatained after needle injection), whereas the expressionof an unmodified reference RNA (R2652) is increased merely by a factorof 2.00. In this example, the expression increase achieved bymodification of the RNA as described herein is 4-fold with respect to anunmodified reference RNA.

TABLE 2 Effect of jet injection using different modified RNAs Effect ofjet injection PpLuc(nat)-A30 R2652 2.00 PpLuc(GC)-muag-A64-C30-HistoneSL8.23 R1265 PpLuc(GC)-A64-C30-HistoneSL R2462 6.25 PpLuc(GC)-muag-A64-C30R1256 3.02 PpLuc(nat)-muag-A64-C30-HistoneSL 2.86 R23932. Induction of a Humoral Immune Response by the RAV-G mRNA Vaccine inHumans

Immunization

Preliminary results obtained in an ongoing clinical trial (phase I)demonstrate safety as well as efficacy of the vaccine according to theinvention. In the clinical study, human volunteers were intradermallyinjected via jet injection using a Tropis device on day 0, 7 and 28 withthe RAV-G mRNA vaccine R2403. The mRNA was prepared as described inExample 1 herein, i.e. mRNA complexed with protamine in a ratio of 2:1(w/w) was mixed with an equal amount of free mRNA. On each of the threevaccination days, 80 μg of mRNA were administered.

In order to assess the safety profile of the vaccine according to theinvention, subjects were monitored after administration (vital signs,vaccination site tolerability assessments, hematologic analysis afterthe second and third injection). The preliminary results obtained in theongoing clinical study suggest that immunization with the mRNA accordingto the invention is well-tolerated in humans.

The efficacy of the immunization was analysed by determination of virusneutralizing titers (VNT) in sera from six subjects. To this end, bloodsamples were collected on day 0 as baseline and on day 42. Sera wereanalysed for virus neutralizing antibodies in the fluorescent antibodyvirus neutralisation (FAVN) test as described below.

Virus Neutralization Test

Detection of the virus neutralizing antibody response (specific B-cellimmune response) was carried out by a virus neutralisation assay. Theresult of that assay is referred to as virus neutralization titer (VNT).According to WHO standards, an antibody titer is considered protectiveif the respective VNT is at least 0.5 IU/ml. The sera obtained asdescribed above were used in a fluorescent antibody virus neutralisation(FAVN) test using the cell culture adapted challenge virus strain (CVS)of rabies virus as recommended by the OIE (World Organisation for AnimalHealth) and first described in Cliquet F., Aubert M. & Sagne L. (1998);J. Immunol. Methods, 212, 79-87. Shortly, heat inactivated sera will betested as quadruplicates in serial two-fold dilutions as quadruplicatesfor their potential to neutralise 100 TCID₅₀ (tissue culture infectiousdoses 50%) of CVS in 50 l of volume. Therefore sera dilutions areincubated with virus for 1 hour at 37° C. (in humid incubator with 5%CO₂) and subsequently trypsinized BHK-21 cells are added (4×10⁵cells/ml; 50 l per well). Infected cell cultures are incubated for 48hours in humid incubator at 37° C. and 5% CO₂. Infection of cells isanalysed after fixation of cells using 80% acetone at room temperatureusing FITC anti-rabies conjugate. Plates were washed twice using PBS andexcess of PBS was removed. Cell cultures are scored positive or negativefor the presence of rabies virus. Negative scored cells in sera treatedwells represent neutralization of rabies virus. Each FAVN tests includesWHO or OIE standard serum (positive reference serum) that serves asreference for standardisation of the assay. Neutralization activity oftest sera is calculated with reference to the standard serum provided bythe WHO and displayed as International Units/ml (IU/ml). The results aresummarized in Table 3.

Results:

TABLE 3 Virus neutralizing titers after immunization of human subjectsVirus neutralizing Subject no. titer (VNT; IU/ml) 1 4.0 2 0.7 3 0.2 40.7 5 1.4 6 0.5

In five out of six subjects (subject no. 1, 2, 4, 5 and 6), a virusneutralizing titer of at least 0.5 IU/ml was detected on day 42.According to the WHO standard, a protective antibody response has thusbeen achieved in these subjects, demonstrating the efficacy of theimmunization with the mRNA according to the invention.

Conclusion:

According to preliminary results from the ongoing clinical trial, theuse of the mRNA according to the invention for immunization of humansubjects has a favourable safety profile. The efficacy of the approachhas been demonstrated by these preliminary studies with a protectiveantibody response (VNT≥0.5 IU/ml) achieved on day 42 in five out of sixinvestigated subjects.

3. Expression of EPO In Vivo

In order to determine erythropoetin expression in vivo, guinea pigs wereintradermally injected with naked mRNA either by conventional needleinjection or by jet injection (3 animals per group, each receiving 3injections of 80 ag of RNA per injection site). After 24 hours, bloodwas sampled from the saphenous vein. Erythropoetin levels in serum weredetermined by ELISA (Mouse Erythropoetin Quantikine ELISA Kit, R&DSystems).

Results: Expression of EPO encoded by a modified mRNA(HSD17B4-EPO(GC)-albumin7-A64-C30-histoneSL (R3135)) is stronglyenhanced in guinea pigs by jet injection compared to conventionalintradermal needle injection (see FIG. 13).

Unexpectedly, expression of EPO from a modified mRNA(HSD17B4-EPO(GC)-albumin7-A64-C30-histoneSL (R3135)) is significantlymore enhanced by jet injection than the expression of EPO from anunmodified reference mRNA (EPO(wt)-A30 (R3513)) (see FIG. 13).

4. Comparison of Conventional and Jet Injection of the RSV-F mRNAVaccine for the Induction of a Humoral Immune Response in Guinea PigsPreparation of DNA and mRNA Constructs:

For the present Examples, DNA sequences encoding the RSV-F protein ofthe RSV long strain (ATCC VR-26) were prepared and used for subsequentin vitro transcription reactions.

Fusion protein F of the RSV strain ATCC VR-26 long (Amino acid sequenceaccording to SEQ ID No. 63):

MELPILKANA ITTILAAVTF CFASSQNITE EFYQSTCSAV SKGYLSALRT GWYTSVITIELSNIKENKCN GTDAKVKLIN QELDKYKNAV TELQLLMQST TAANNRARRE LPRFMNYTLNNTKKTNVTLS KKRKRRFLGF LLGVGSAIAS GIAVSKVLHL EGEVNKIKSA LLSTNKAVVSLSNGVSVLTS KVLDLKNYID KQLLPIVNKQ SCRISNIETV IEFQQKNNRL LEITREFSVNAGVTTPVSTY MLTNSELLSL INDMPITNDQ KKLMSNNVQI VRQQSYSIMS IIKEEVLAYVVQLPLYGVID TPCWKLHTSP LCTTNTKEGS NICLTRTDRG WYCDNAGSVS FFPQAETCKVQSNRVFCDTM NSLTLPSEVN LCNVDIFNPK YDCKIMTSKT DVSSSVITSL GAIVSCYGKTKCTASNKNRG IIKTFSNGCD YVSNKGVDTV SVGNTLYYVN KQEGKSLYVK GEPIINFYDPLVFPSDEFDA SISQVNEKIN QSLAFIRKSD ELLHHVNAGK STTNIMITTI IIVIIVILLSLIAVGLLLYC KARSTPVTLS KDQLSGINNI AFSN

According to a first preparation, the DNA sequences encoding the abovementioned mRNAs were prepared. The DNA construct encoding RNA sequenceR2510 (SEQ ID NO: 64) was prepared by introducing a 5′-TOP-UTR derivedfrom the ribosomal protein 32L according to SEQ ID No. 55, modifying thewild type coding sequence by introducing a GC-optimized sequence forstabilization, followed by a stabilizing sequence derived from thealbumin-3′-UTR (albumin7 according to SEQ ID No. 58), a stretch of 64adenosines (poly(A)-sequence), a stretch of 30 cytosines(poly(C)-sequence), and a histone stem loop according to SEQ ID No. 44.In FIG. 14 the sequence of the corresponding mRNA (R2510; SEQ ID NO: 64)is shown.

In Vitro Transcription:

The respective DNA plasmids prepared according to paragraph 1 weretranscribed in vitro using T7 polymerase in the presence of a CAP analog(m⁷GpppG). Subsequently the mRNA was purified using PureMessenger®(CureVac, Tiibingen, Germany; WO2008/077592A1).

Reagents:

Complexation Reagent: protamine

Preparation of the Vaccine:

The mRNA was complexed with protamine by addition of protamine to themRNA in the ratio (1:2) (w/w) (adjuvant component). After incubation for10 minutes, the same amount of free mRNA used as antigen-providing RNAwas added.

RSV-F long vaccine (R2510): comprising an adjuvant component consistingof mRNA coding for RSV F protein long (R2510) according to SEQ ID NO. 63complexed with protamine in a ratio of 2:1 (w/w) and theantigen-providing free mRNA coding for RSV F protein long (R2510)according to SEQ ID NO. 63 (ratio 1:1; complexed RNA:free RNA).

Immunization

On day zero, female guinea pigs (n=8/group) were intradermally (i.d.)injected with the RSV-F mRNA vaccine as described above (80 ag ofR2510/mouse/vaccination day) either 1×100 al with conventional needleinjection (i.d.), 4×25 μl with needle injection (i.d.), or 1×100 μl withjet injection (i.d.) as shown in Table 4. A control group (n=2) wasneedle-injected intramuscularly (i.m.) with 20 ag of inactivated RSVlong (2×50 al). The inactivated “Respiratory Syncytial Virus Antigen”(inactivated RSV long) was purchased from the INSTITUT VIRION/SERIONGmbH-SERION IMMUNDIAGNOSTICA GmbH. The inactivated virus was diluted insterile PBS, so that a final concentration of 0.2 ag/L was achieved. Allanimals received boost injections on days 14 and 28. Blood samples werecollected on day −3 (three days before the first vaccination) and ondays 7, 21 and 42 for the determination of anti-RSV F antibody titers.

TABLE 4 Animal groups Group Strain sex No. mice Volume route Vaccinedose Vaccination day Bleeding day 1 Guinea Pigs, 8 1 × 100 μl, R2510 80μg 0, 14, 28 −3, 7, 21 ,42 female i.d., conventional 2 Guinea Pigs, 8 4× 25 μl, R2510 80 μg 0, 14, 28 −3, 7, 21 ,42 female i.d., conventional 3Guinea Pigs, 8 1 × 100 μl, R2510 80 μg 0, 14, 28 −3, 7, 21 ,42 femalei.d., jet injection 4 Guinea Pigs, 2 2 × 50 μl, Inactivated RSV 0, 14,28 −3, 7, 21 ,42 female i.m., conventional long 20 μg

Determination of Anti-RSV F Protein Antibodies by ELISA

ELISA plates were coated with inactivated RSV-LONG (Virion/Serion, finalconcentration 5 ag/mL) (Sino Biological Inc.). Coated plates wereincubated with guinea pig serum using indicated dilutions (1:50 to1:3906250 in 5× dilution steps) and binding of specific antibodies tothe F protein was detected using biotinylated isotype specificanti-guinea pig IgG and IgG2a antibodies in combination withstreptavidin-HRP (horse radish peroxidase) with 2,2′-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS)substrate.

RSV Neutralizing Antibody Titers (VNTs)

Sera were analysed by determining virus neutralization titers (VNTs).Briefly, sera samples were diluted 1:10 with Eagle's Minimum EssentialMedium (EMEM), heat inactivated and serially diluted further 1:4.Diluted sera samples were incubated with RSV (25-50 PFU) for one hour atroom temperature and inoculated in duplicates onto confluent HEp-2 cellmonolayers in 24 well plates. After one hour incubation at 37° C. in a5% CO₂ incubator, the wells were overlayed with 0.75% Methylcellulosemedium. After 4 days of incubation, the overlay was removed and thecells were fixed with 0.1% crystal violet stain for one hour and thenrinsed and air dried. The corresponding reciprocal neutralizing antibodytiters were determined at the 60% reduction end-point of the viruscontrol.

Results

As shown in FIG. 15, the RSV-F mRNA vaccine already induced anti-Fprotein antibodies of the IgG1 subclass (A) and the IgG2a subclass (B)on day 21 (one week after the first boost vaccination on day 14) whenthe vaccine was administered by jet injection (1×100 al). Comparableantibody titers were only reached on day 42 (two weeks after the secondboost vaccination on day 28) when the vaccine was administered byconventional needle injection (4×25 al). Thus the RSV-F mRNA vaccineadministered by jet injection induced a humoral immune response againstthe RSV-F protein with a faster kinetic compared to administration byconventional needle injection.

As shown in FIG. 16, significant RSV neutralization titers were onlymeasured on day 42 when the vaccine was administered by jet injection(100 al). Thus the vaccination with jet injection leads to a fasteronset of immune responses than conventional needle injection.

1. A modified RNA comprising at least one open reading frame andcomprising at least one modification, which increases the expression ofthe encoded peptide or protein, for use in medical treatment, whereinthe modified RNA is administered by jet injection.
 2. The modified RNAfor use according to claim 1, wherein the RNA is administeredintradermally.
 3. The modified RNA for use according to claim 1, whereinthe RNA is administered intramuscularly.
 4. The modified RNA for useaccording to claim 1, wherein the RNA is administered subcutaneously. 5.The modified RNA for use according to any of claims 1 to 4, wherein themodified RNA is characterized by an increased expression of the encodedpeptide or protein in comparison to an unmodified reference RNA.
 6. Themodified RNA for use according to any of claims 1 to 5, wherein theexpression of a peptide or protein encoded by the modified RNA isincreased by at least 5-fold in comparison to a reference, where thesame modified RNA is administered by means other than jet injection. 7.The modified RNA for use according to any of claims 1 to 6, wherein themodified RNA encodes at least one antigen, a therapeutic protein or anantibody.
 8. The modified RNA for use according to any of claims 1 to 7,wherein the at least one modification is a modification selected fromthe group consisting of modification of the G/C content, codonoptimization, UTR modification, Poly(A) tail with more than 30 adenosinenucleotides, Poly(C) sequence, 5′-CAP structure except of m7GpppN,histone-stem-loop sequence and a chemically modified nucleotide.
 9. Themodified RNA for use according to claim 8, wherein the at least onemodification is a chemically modified ARCA-CAP.
 10. The modified RNA foruse according to claim 9, wherein the ARCA-CAP is modified withphosphorothioate.
 11. The modified RNA for use according to claim 8,wherein the at least one modification is a UTR modification and the UTRis modified by introducing a UTR from a stable RNA.
 12. The modified RNAfor use according to claim 11, wherein the stable RNA is selected fromalpha globin UTR and beta globin UTR.
 13. The modified RNA for useaccording to claim 8, wherein the at least one modification is an UTRmodification and the UTR is modified by introducing a 5′-UTR from a TOPgene.
 14. The modified RNA for use according to claim 8, wherein thechemically modified nucleotide is selected frompseudouridine-5′-triphosphate and 5-methylcytidine-5′-triphosphate. 15.The modified RNA for use according to claim 8, wherein the modified RNAcomprises the following modifications: a modification of the G/Ccontent, at least one UTR modification, a poly(A) tail with more than 30adenosine nucleotides, and a histone stem-loop sequence.
 16. Themodified RNA for use according to claim 15, wherein the histonestem-loop sequence is a nucleic acid sequence according to SEQ ID No.44.
 17. The modified RNA for use according to claim 15 or 16, whereinthe modified RNA additionally comprises a m7GpppN 5′-CAP.
 18. Themodified RNA for use according to any of claims 15-17, wherein thepoly(A) tail comprises more than 50 adenosine nucleotides.
 19. Themodified RNA for use according to any of claims 15-18, wherein thepoly(A) tail consist of 64 adenosine nucleotides.
 20. A pharmaceuticalcomposition comprising a modified RNA comprising at least onemodification, which increases the expression of the encoded peptide orprotein, and comprising at least one open reading frame coding for atleast one therapeutic peptide or protein, wherein the pharmaceuticalcomposition is administered by jet injection, the pharmaceuticalcomposition further comprising at least one suitable pharmaceuticalexcipient.
 21. The modified RNA as defined according to any one ofclaims 1 to 19 or the pharmaceutical composition as defined according toclaim 20 for use in the treatment of prostate cancer.
 22. A method forenhancing the localized expression of an RNA-encoded peptide or proteinin the dermis or muscle of a mammal comprising administering by jetinjection a modified RNA comprising at least one modification, whichincreases the expression of the encoded peptide or protein, andcomprising at least one open reading frame coding for said peptide orprotein.
 23. The method of claim 22, wherein the RNA-encoded protein isan antigen, therapeutic protein or an antibody.
 24. A method oftreatment comprising administering a modified RNA comprising at leastone modification which increases the expression of the encoded peptideor protein and comprising at least one open reading frame coding for atleast one therapeutic peptide or protein by jet injection into a subjectin need thereof.
 25. The method according to claim 24, wherein themodified RNA is injected intradermally, intramuscularly orsubcutaneously.
 26. Use of a modified RNA for the manufacture of amedicament in the treatment of prostate cancer.
 27. A kit of partscomprising a modified RNA comprising at least one modification whichincreases the expression of the encoded peptide or protein andcomprising at least one open reading frame for use in a in medicaltreatment, wherein the modified RNA is administered by jet injectionaccording to claims 1 to 24, in particular according to claims 1 to 19,or the pharmaceutical composition according to claim 20 or
 25. 28. Thekit according to claim 27, further comprising instructions for use, anadjuvant, a means for administration of the pharmaceutical composition,a pharmaceutically acceptable carrier and/or a pharmaceuticallyacceptable solution for dissolution or dilution of the modified RNA orthe pharmaceutical composition.